The influence of Acetobacter pomorum bacteria on the developmental progression of Drosophila suzukii via gluconic acid secretion.

Insects are rich in various microorganisms, which play diverse roles in affecting host biology. Although most Drosophila species prefer rotten fruits, the agricultural pest Drosophila suzukii attacks ripening fruits before they are harvested. We have reported that the microbiota has positive and negative impacts on the agricultural pest D. suzukii on nutrient-poor and -rich diets, respectively. On nutrient-poor diets, microbes provide protein to facilitate larval development. But how they impede D. suzukii development on nutrient-rich diets is unknown. Here we report that Acetobacter pomorum (Apo), a commensal bacterium in many Drosophila species and rotting fruit, has several detrimental effects in D. suzukii. Feeding D. suzukii larvae nutrient-rich diets containing live Apo significantly delayed larval development and reduced the body weight of emerged adults. Apo induced larval immune responses and downregulated genes of digestion and juvenile hormone metabolism. Knockdown of these genes in germ-free larvae reproduced Apo-like weakened phenotypes. Apo was confirmed to secrete substantial amounts of gluconic acid. Adding gluconic acid to the D. suzukii larval diet hindered larval growth and decreased adult body weight. Moreover, the dose of gluconic acid that adversely affected D. suzukii did not negatively affect Drosophila melanogaster, suggesting that D. suzukii is less tolerant to acid than D. melanogaster. Taken together, these findings indicate that D. suzukii is negatively affected by gluconic acid, which may explain why it prefers ripening fruit over Apo-rich rotting fruit. These results show an insect's tolerance to microbes can influence its ecological niche.

Revealing the phosphate-solubilizing characteristics and mechanisms of the plant growth-promoting bacterium Agrobacterium deltaense C1.

AIMS: This study explores the phosphate (Pi)-solubilizing characteristics and mechanisms of a novel phosphate-solubilizing bacterium, Agrobacterium deltaense C1 (C1 hereafter). METHODS AND RESULTS: The growth-promoting effects of C1 were investigated by gnotobiotic experiments, and the Pi-solubilizing mechanism was revealed by extracellular metabolomics, liquid chromatography analysis, and reverse transcription quantitative polymerase chain reaction. Results showed that C1 significantly increased Arabidopsis biomass and total phosphorus (P) content under P deficiency. Under Ca3(PO4)2 condition, the presence of C1 resulted in a significant and negative correlation between available P content and medium pH changes, implying that Pi dissolution occurs through acid release. Metabolomics revealed C1's ability to release 99 organic acids, with gluconic acid (GA), citric acid, and α-ketoglutaric acid contributing 64.86%, 9.58%, and 0.94%, respectively, to Pi solubilization. These acids were significantly induced by P deficiency. Moreover, C1's Pi solubilization may remain significant even in the presence of available P, as evidenced by substantial pH reduction and high gcd gene expression. Additionally, C1 produced over 10 plant growth-promoting substances. CONCLUSIONS: C1 dissolves Pi primarily by releasing GA, which enhances plant growth under P deficiency. Notably, its Pi solubilization effect is not significantly limited by available Pi.

Dilute gluconic acid pretreatment and fermentation of wheat straw to ethanol.

Gluconic acid's potential as a wheat straw pretreatment agent was studied at different concentrations (0.125-1 M) and temperatures (160-190 °C) for 30 min, followed by enzymatic hydrolysis. 0.125 M gluconic acid, 170 °C, yielded the highest xylose output, while 0.5 M gluconic acid at 190 °C yielded the best glucose yield. A fraction of gluconic acid decomposed during pretreatment. Detoxified hemicellulose hydrolysate from 0.125 M gluconate at 170 °C for 60 min showed promise for ethanol production. The gluconate contained in the detoxified hemicellulose hydrolysate can be fermented to ethanol along with other hemicellulose sugars present by Escherichia coli SL100. The ethanol yield from gluconate and sugars was about 90.4 ± 1.8%. The pretreated solids can be effectively converted to ethanol by Saccharomyces cerevisiae D5A via simultaneous saccharification and fermentation with the cellulase and ß-glucosidase addition. The ethanol yield achieved was 92.8 ± 2.0% of the theoretical maximum. The cellulose conversion was about 70.8 ± 0.8%.

The role of proton excreted by Advenella kashmirensis DF12 during ammonium assimilation in phosphate solubilization.

Phosphate-solubilizing bacteria (PSB) can solubilize soil fixed phosphorus (P) to plant available forms. In previous studies, the mechanisms of inorganic phosphate solubilization by PSB mostly focused on the acidolysis of organic acids. Here we screened a highly efficient PSB, Advenella kashmirensis DF12, with the maximum P solubilization of 590 mg L- 1 at 6 days. In addition to its P solubilizing ability, DF12 also showed a tolerance to pH from 5 to 10 and a nitrogen fixation potential. The multiple functions of DF12 and its wide adaptability to various environmental conditions make it a promising biofertilizer candidate. The combined analysis of extracellular metabolites and intracellular metabolome data revealed that the production of organic acid (mainly gluconic acid) is not the only mechanism of P solubilized by DF12, the solubilized P content was not correlated with the gluconic acid concentration but was in a highly significant positive correlation with proton concentration, extrusion of proton during NH4+ assimilation plays a key role in phosphate solubilization. Moreover, the contribution of NH4+ assimilation to phosphorus solubilization is generally present in PSB. Therefore, we proposed that applying ammonium fertilizer in P-deficient soil is more appropriate, it can not only supplement nitrogen fertilizer, but also enhance P use efficiency, which contributes to worldwide fertilizer use reduction and efficiency improvement.

Compositional evolution for mixed aerosols containing gluconic acid and typical nitrate and the effect of multiply factors on hygroscopicity.

The aging process of atmospheric aerosols usually leads to a mixture of inorganic salts and organic compounds of anthropogenic origin. In organic compounds, polyhydroxy organic acids are important components, however, the study on composition and hygroscopic properties of the mixture containing inorganics and polyhydroxy organic acids is scanty. In this study, gluconic acid, the proxy of polyhydroxy organic acids, is mixed with the representative nitrate (Mg(NO3)2, Ca(NO3)2) to form aerosols. ATR-FTIR and optical microscopy are employed to study the component changes and hygroscopicity as a function of relative humidity. As relative humidity fluctuates, the FTIR-ATR spectra display that the internal mixed gluconic acid (CH2(CH)4(OH)5COOH) and nitrate can react to release acidic gases, forming relevant gluconate and further affecting the hygroscopicity. The specific presentation is particles cannot be recovered to their original size after the dehydration-hydration process and there will be some disparities in GF for mixed particles. For the gluconic acid-Ca(NO3)2/Mg(NO3)2 mixtures with molar ratios of 1:1, higher degree of reaction resulting in the production of large amounts of gluconate should be responsible to the lower hygroscopicity compared to ZSR model. For 1:2 gluconic acid-nitrate mixed systems (with higher nitrate content), the hygroscopicity of mixtures are higher than the ZSR prediction. A possible reason could be 'salt-promoting effect' on the organic fractions of the surplus inorganic salt in the mixture. These data can improve the chemical composition list evaluation, in turn hygroscopic properties and phase state of atmospheric aerosol, and then the climate effect.

Ulva rigida Valorization into Poly(3-hydroxybutyrate), Organic Acids and Functional Ingredients.

Halomonas elongata 1H9T is a moderate halophilic strain able to produce poly(3-hydroxybutyrate) (P(3HB)), a biodegradable plastic, and gluconic acid, a valuable organic acid with wide industrial applications. In this work, the green alga Ulva rigida was used as platform to produce cultivation substrates for microbial conversion as well as functional ingredients, targeting its full valorization. The liquor obtained by autohydrolysis presented the highest concentration of oligosaccharides and protein, being an interesting feedstock to produce functional ingredients. The acid and/or enzymatic hydrolysis liquors are adequate as substrates for microbial processes. Shake flask assays with H. elongata revealed that the N-rich liquor produced after acidic treatment was the best suited for cell growth while the N-poor liquor produced by the enzymatic treatment of acid-pretreated algae residues produced the highest P(3HB) titers of 4.4 g/L. These hydrolysates were used in fed-batch cultivations as carbon and protein sources for the co-production of gluconic acid and polymer achieving titers of 123.2 g/L and 7.2 g/L, respectively. Besides gluconic acid, the Krebs cycle intermediate 2-oxoglutaric acid, also called alpha-ketoglutaric acid (KGA), was produced. Therefore, the co-production of P(3HB) and acids may be of considerable interest as an algal biorefinery valorization strategy.

Efficient aerobic fermentation of gluconic acid by high tension oxygen supply strategy with reusable Gluconobacter oxydans HG19 cells.

Gluconic acid is a widely used food and beverage additive, but its production suffers from low efficiency and high cost. In this study, a preferable gluconic acid biosynthesis method without repeated seed culture was proposed and developed using the superior performance of Gluconobacter oxydans. A high oxygen atmosphere satisfying the demand of bio-oxidation increased the productivity of gluconic acid up to ~ 32 g/L/h and the accumulation up to ~ 420 g/L within 24 h of fed-batch fermentation. However, the productivity remarkably decreased when the gluconic acid content was over 350 g/L. Therefore, a continuous fermentation was designed, which in combination with 5 runs of fed-batch fermentation resulted in the final production of 1700 g gluconic acid from 1750 g glucose within 60 h in a 3 L bioreactor. The results suggest that the validity of this model and can enable cost-competitive gluconic acid production in the industry.

2-Keto-D-gluconic acid and prodigiosin producing by a Serratia marcescens.

Microbial fermentation has become the main method to produce target compound. In this study, a 2-Keto-D-gluconic acid (2-KGA) producing mutant strain was obtained by mutation with rational screening methods. Meanwhile, prodigiosin was produced when the nitrogen source in the medium was changed to peptone and its fermentation conditions were evaluated to achieve high-efficient accumulation. The mutant strain SDSPY-136 was firstly identified as Serratia marcescens by morphological observation and 16S rDNA sequencing. The 2-KGA synthetic capacity of S. marcescens SDSPY-136 was evaluated by shake fermentation with 110 g/L glucose as substrates. For fermentation, 2-KGA yield, conversation rate and purity of SDSPY-136 reached 104.60 g/L, 95.10%, 99.11% in 72 h. The red pigment was extracted from the fermentation broth using acidic methanol and identified as prodigiosin by FT-IR. The optimal conditions were as follows: glycerol 20 g/L, peptone 20 g/L, MgSO415 g/L, pH 6.0, a 2% (v/v) inoculum, 30 °C and 200 rpm of shaking culture. Eventually, prodigiosin reached a yield of 9.89 g/Lafter shake culturing for 50 h under this condition. The mutant S. marcescens SDSPY-136 was shown to be promising for 2-KGA and prodigiosin production and a suitable object for prodigiosin metabolism research of S. marcescens.

Studies on leaching characteristics of electronic waste for metal recovery using inorganic and organic acids and base.

In this paper, we report leaching of precious and scattered metals such as gold (Au), copper (Cu), nickel (Ni), zinc (Zn), iron (Fe), and lead (Pb) from printed circuit boards of scrap mobile phones by hydrometallurgical process using inorganic acid, organic acid and base. The amount of metals leached by different leachants are quantified using atomic absorption spectroscopy. Among various inorganic acids, aqua regia (mixture of nitric acid (HNO3) and hydrochloric acid) is found to be the strongest leachant for most of the metals such as Zn (2.04 wt %), Fe (17.90 wt %), Ni (0.66 wt %), Pb (5.86 wt %) and Au (0.04 wt %). The basic leachant, ammonium thiosulphate is found to be very effective in leaching of Au (0.03125 wt %). The dissolution of Cu in HNO3 gives the highest amount of Cu in the solvent, that is, ∼ 7.52 wt %. The metallic phases present in the electronic waste before and after leaching are identified by X-ray diffraction analysis. The microscopic structure has been studied using a scanning electron microscope which depicts erosion of the structure after leaching.

A simple enzymatic assay for the quantification of C1-specific cellulose oxidation by lytic polysaccharide monooxygenases.

OBJECTIVE: The development of an enzymatic assay for the specific quantification of the C1-oxidation product, i.e. gluconic acid of cellulose active lytic polysaccharide monooxygenases (LPMOs). RESULTS: In combination with a ß-glucosidase, the spectrophotometrical assay can reliably quantify the specific C1- oxidation product of LPMOs acting on cellulose. It is applicable for a pure cellulose model substrate as well as lignocellulosic biomass. The enzymatic assay compares well with the quantification performed by HPAEC-PAD. In addition, we show that simple boiling is not sufficient to inactivate LPMOs and we suggest to apply a metal chelator in addition to boiling or to drastically increase pH for proper inactivation. CONCLUSIONS: We conclude that the versatility of this simple enzymatic assay makes it useful in a wide range of experiments in basic and applied LPMO research and without the need for expensive instrumentation, e.g. HPAEC-PAD.

The NMR signature of gluconoylation: a frequent N-terminal modification of isotope-labeled proteins.

N-terminal gluconoylation is a moderately widespread modification in recombinant proteins expressed in Escherichia coli, in particular in proteins bearing an N-terminal histidine-tag. This post-translational modification has been investigated mainly by mass spectrometry. Although its NMR signals must have been observed earlier in spectra of 13C/15N labeled proteins, their chemical shifts were not yet reported. Here we present the complete 1H and 13C chemical shift assignment of the N-terminal gluconoyl post-translational modification, based on a selection of His-tagged protein constructs (CCL2, hnRNP A1 and Lin28) starting with Met-Gly-...-(His)6. In addition, we show that the modification can hydrolyze over time, resulting in a free N-terminus and gluconate. This leads to the disappearance of the gluconoyl signals and the appearance of gluconate signals during the NMR measurements. The chemical shifts presented here can now be used as a reference for the identification of gluconoylation in recombinant proteins, in particular when isotopically labeled.

Succinate irrepressible periplasmic glucose dehydrogenase of Rhizobium sp. Td3 and SN1 contributes to its phosphate solubilization ability.

Td3 and SN1 are phosphate-solubilizing nodule rhizobia of Cajanus cajan and Sesbania rostrata, respectively. They solubilized 423 µg/mL and 428 µg/mL phosphate from tricalcium phosphate through the secretion of 19.2 mM and 29.6 mM gluconic acid, respectively, when grown in 100 mM glucose. However, 90% and 80% reduction in phosphate solubilization coupled to the production of 40 mM (Td3) and 28.2 mM (SN1) gluconic acid was observed when the two isolates were grown in 50 mM succinate + 50 mM glucose. Our results illustrate the role of succinate irrepressible glucose dehydrogenase (gcd) in phosphate solubilization and the role of succinate: proton symport in succinate-mediated repression of phosphate solubilization in these rhizobia. Calcium ion supplementation reduced the 88% and 72% repression in P solubilization to 18% and 9% in Td3 and SN1, respectively, coupled to a reduction in media pH from 6.8 to 4.9 in Td3 and 6.3 to 4.8 in SN1. Hence, repression had no genetic basis and is purely due to the biochemical interplay of protons and other cations.

Modified semi-continuous fermentation for resuscitating nongrowing cells during high-temperature gluconic acid production by Acetobacter senegalensis.

AIMS: The formation of metabolically inactive and nongrowing cells is an inevitable by-product of intensive fermentation. This study investigated whether co-feeding can be used to resuscitate nongrowing Acetobacter senegalensis cells to enable them to produce gluconic acid in successive fermentation runs at 38°C. METHODS AND RESULTS: In the first fermentation cycle, 75 g l-1 of glucose were converted to gluconic acid. Subsequently, however, stationary-phase cells were unable to initiate a new fermentation cycle. The majority of stationary-phase cells (97%) were nonculturable on glucose at 38°C. In addition, 54 and 41% of cells contained non-active cellular dehydrogenases and a compromised cell envelope respectively. Co-feeding stationary-phase cells with a mixture of ethanol, glucose and acetic acid for 7 h enabled these cells to grow on 75 g l-1 of glucose and produce gluconic acid. Additionally, 74% of cells contained active forms of cellular dehydrogenases after 7 h of co-feeding. However, co-feeding did not improve cell envelope integrity. Quantification of cellular NAD content showed that stationary-phase cells contained moderately reduced levels of total NAD (NADt) as compared with exponential-phase cells. Interestingly, the analysis of stationary-phase cells showed that co-feeding resulted in higher levels of NADt and NADH, suggesting that the regeneration of NADH is one of the limiting factors of glucose consumption. Expression of catalase and superoxide dismutase was increased in stationary-phase cells, but analysis of protein carbonylation and lipid peroxidation did not confirm an extensive oxidative stress. CONCLUSIONS: Co-feeding with favourable nutrients may enable resuscitation of cells and utilization of less-favourable carbon sources in successive cycles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposed a unique method for resuscitation of nongrowing cells during high-temperature fermentation. By applying this method, cells can be used for consecutive fermentation cycles.

Genome sequencing of Aureobasidium pullulans P25 and overexpression of a glucose oxidase gene for hyper-production of Ca2+-gluconic acid.

Gluconic acid (GA) has many applications such as in the food and pharmaceutical industry. Aureobasidium pullulans P25 strain is able to produce high levels of Ca2+-GA. The genome length, GC content and the gene number of this yeast were found to be 30.97 Mb, 50.28% and 10,922, respectively. The pathways for gluconic acid biosynthesis were annotated. Glucose oxidase (Gox) sequences from different strains of A. pullulans were highly similar but were distinct from those of other fungi. The glucose oxidase had two FAD binding sites and a signal sequence. Deletion of the GOX gene resulted in a strain that showed no Gox activity and that was unable to produce Ca2+-GA. Overexpression of the GOX gene in strain P25 generated strain GA-6 that produced 200.2 ± 2.3 Ca2+-GA g/l and 2480 U/mg of Gox activity. The productivity of Ca2+-GA was 2.78 g/l/h and the yield was 1.1 g/g.

Postruminal infusion of calcium gluconate increases milk fat production and alters fecal volatile fatty acid profile in lactating dairy cows.

Gluconic acid is a carboxylic acid naturally occurring in plants and honey. In nonruminant animals, gluconic acid has been shown to increase gastrointestinal butyrate concentrations and improve growth performance, but a ruminant application remains undescribed. This experiment examined the effects of postruminal calcium gluconate (CaG) on milk production, fecal volatile fatty acid concentrations, and plasma metabolite concentrations in lactating dairy cows. Six rumen cannulated multiparous Holstein cows (60 ± 6 d in milk) were randomly assigned to 6 treatment sequences within a 6 × 6 Latin square design in which each experimental period consisted of 5 d of continuous postruminal infusion followed by a 2 d wash-out period. Test treatments included a negative control (CON; 0.90% NaCl wt/vol), positive control (Na-butyrate, 135 g/d), and 4 doses of CaG (44, 93, 140, and 187 g/d). Cows received a total mixed ration (31% corn silage, 28% alfalfa silage, 5% hay, 36% concentrate) with dry matter intake fixed (25.3 ± 1.7 kg/d) throughout the experiment. On d 5 of each infusion period, samples of milk, feces, and blood were collected from each animal. Calcium gluconate treatments increased milk fat concentration, and a tendency was observed for increased milk fat yield and energy-corrected milk yield above levels achieved by CON, with maximal treatment responses of 4.43% (CON 3.81%), 2.089 kg/d (CON 1.760 kg/d), and 51.8 kg/d (CON 47.1 kg/d), respectively. Concentrations of iso-butyric acid in feces were greater in cows infused with CaG (13.3 µmol/g) treatments compared with CON (9.7 µmol/g). Arterial concentrations of glucose and nonesterified fatty acids were lower (glucose: CaG 2.98 mmol/L, CON 3.29 mmol/L and nonesterified fatty acids: CaG 0.130 mmol/L vs. 0.148 mmol/L) and ß-hydroxybutyrate higher (CaG 1.703 vs. CON 0.812) in cows infused with CaG than CON. Together, these results suggest that postruminal infusion of CaG may alter metabolic mechanisms to support a milk fat production response.

Modeling and simulation of enzymatic gluconic acid production using immobilized enzyme and CSTR-PFTR circulation reaction system.

OBJECTIVES: Production of gluconic acid by using immobilized enzyme and continuous stirred tank reactor-plug flow tubular reactor (CSTR-PFTR) circulation reaction system. RESULTS: A production system is constructed for gluconic acid production, which consists of a continuous stirred tank reactor (CSTR) for pH control and liquid storage and a plug flow tubular reactor (PFTR) filled with immobilized glucose oxidase (GOD) for gluconic acid production. Mathematical model is developed for this production system and simulation is made for the enzymatic reaction process. The pH inhibition effect on GOD is modeled by using a bell-type curve. CONCLUSIONS: Gluconic acid can be efficiently produced by using the reaction system and the mathematical model developed for this system can simulate and predict the process well.

Synthesis of orange-red emissive carbon dots for fluorometric enzymatic determination of glucose.

The authors describe a method for the preparation of orange-red emissive carbon dots (CDs) with excitation/emission peaks at 520/582 nm. The CDs were hydrothermally prepared by a one-pot strategy from trimesic acid and 4-aminoacetanilide. The fluorescence of the CDs is strongly quenched by hydrogen peroxide. The oxidation of glucose by glucose oxidase (GOx) produces H2O2 that quenches the fluorescence via static quenching. Based on this phenomenon, a fluorometric method was established for the determination of glucose. Under the optimum conditions, response is linear in the 0.5 to 100 µM glucose concentration range, with a 0.33 µM limit of detection. The method is selective for glucose over its analogues and was successfully applied to the determination of glucose in diluted human serum and in urine from diabetics and healthy individuals. Recoveries from spiked samples range from 98.7 to 102.5%. Graphical abstract (a) One-step synthetic strategy of the CDs; (b) Schematic illustration of the CDs for glucose detection.

Oxygen-enriched fermentation of sodium gluconate by Aspergillus niger and its impact on intracellular metabolic flux distributions.

Different concentrations of oxygen-enriched air were utilized for sodium gluconate (SG) fermentation by Aspergillus niger. The fermentation time shortened from 20 to 15.5 h due to the increase of volumetric oxygen transfer coefficient (KLa) and the formation of more dispersed mycelia when inlet oxygen concentration ascended from 21 to 32%. According to metabolic flux analysis, during the growth phase, extracellular glucose for SG synthesis accounted for 79.0 and 85.3% with air and oxygen-enriched air (25%), respectively, whereas the proportions were 89.4 and 93.0% in the stationary phase. Intracellular glucose consumption decreased in oxygen-enriched fermentation, as cell respiration was more high-efficiently performed. Metabolic profiling indicated that most intermediates in TCA cycle and EMP pathway had smaller pool sizes in oxygen-enriched fermentations. Moreover, the main by-product of citric acid dramatically decreased from 1.36 to 0.34 g L-1 in oxygen-enriched fermentation. And the sodium gluconate yield increased from 0.856 to 0.903 mol mol-1.

Part-2: Analytical Expressions of Concentrations of Glucose, Oxygen, and Gluconic Acid in a Composite Membrane for Closed-Loop Insulin Delivery for the Non-steady State Conditions.

A mathematical model developed by Abdekhodaie and Wu (J Membr Sci 335:21-31, 2009), which describes a dynamic process involving an enzymatic reaction and diffusion of reactants and product inside glucose-sensitive composite membrane has been discussed. This theoretical model depicts a system of non-linear non-steady state reaction diffusion equations. These equations have been solved using new approach of homotopy perturbation method and analytical solutions pertaining to the concentrations of glucose, oxygen, and gluconic acid are derived. These analytical results are compared with the numerical results, and limiting case results for steady state conditions and a good agreement is observed. The influence of various kinetic parameters involved in the model has been presented graphically. Theoretical evaluation of the kinetic parameters like the maximal reaction velocity (V max) and Michaelis-Menten constants for glucose and oxygen (K g and K ox) is also reported. This predicted model is very much useful for designing the glucose-responsive composite membranes for closed-loop insulin delivery.

Influence of carbon sources on the viability and resuscitation of Acetobacter senegalensis during high-temperature gluconic acid fermentation.

Much research has been conducted about different types of fermentation at high temperature, but only a few of them have studied cell viability changes during high-temperature fermentation. In this study, Acetobacter senegalensis, a thermo-tolerant strain, was used for gluconic acid production at 38 °C. The influences of different carbon sources and physicochemical conditions on cell viability and the resuscitation of viable but nonculturable (VBNC) cells formed during fermentation were studied. Based on the obtained results, A. senegalensis could oxidize 95 g l- 1 glucose to gluconate at 38 °C (pH 5.5, yield 83%). However, despite the availability of carbon and nitrogen sources, the specific rates of glucose consumption (qs) and gluconate production (qp) reduced progressively. Interestingly, gradual qs and qp reduction coincided with gradual decrease in cellular dehydrogenase activity, cell envelope integrity, and cell culturability as well as with the formation of VBNC cells. Entry of cells into VBNC state during stationary phase partly stemmed from high fermentation temperature and long-term oxidation of glucose, because just about 48% of VBNC cells formed during stationary phase were resuscitated by supplementing the culture medium with an alternative favorite carbon source (low concentration of ethanol) and/or reducing incubation temperature to 30 °C. This indicates that ethanol, as a favorable carbon source, supports the repair of stressed cells. Since formation of VBNC cells is often inevitable during high-temperature fermentation, using an alternative carbon source together with changing physicochemical conditions may enable the resuscitation of VBNC cells and their use for several production cycles.