Pilot-scale Fermentation Research Articles

Nutritional properties of pilot-scale manufactured single-cell proteins from a Corynebacterium glutamicum

ABSTRACT Single-cell proteins (SCPs) derived from microorganisms are sustainable and standard-manufacturable protein sources. Corynebacterium glutamicum is one of the prospective bacteria for SCP production. To evaluate nutritional properties of C. glutamicum-SCP, pilot-scale fermentation (3,000 L) was performed under standard conditions, resulting in 23 kg of SCP in dry weight. This C. glutamicum-SCP had high protein and low-fat contents (22.4% carbohydrates, 60.9% crude protein, and 3.3% crude fat). Its fatty acid profile comprised 0.25% saturated fatty acids, 0.16% unsaturated fatty acids, no trans fatty acids, and no cholesterol. Its total iron content was 9.78 mg/100 g, of which approximately 14.8% was heme iron (1.45 mg/100 g). The Digestible Indispensable Amino Acid Score (DIAAS) of C. glutamicum-SCP was 102, higher than that of soy protein isolate (90), pea protein concentrate (82), house cricket (89), or yellow mealworm (64). In conclusion, C. glutamicum-SCP can be a promising alternative protein source to replace red meat.

A Genomewide Evolution-Based CRISPR/Cas9 with Donor-Free (GEbCD) for Developing Robust and Productive Industrial Yeast.

Developing more robust and productive industrial yeast is crucial for high-efficiency biomanufacturing. However, the challenges posed by the long time required and the low abundance of mutations generated through genomewide evolutionary engineering hinder the development and optimization of desired hosts for industrial applications. To address these issues, we present a novel solution called the Genomewide Evolution-based CRISPR/Cas with Donor-free (GEbCD) system, in which nonhomologous-end-joining (NHEJ) repair can accelerate the acquisition of highly abundant yeast mutants. Together with modified rad52 of the DNA double-strand break repair in Saccharomyces cerevisiae, a hypermutation host was obtained with a 400-fold enhanced mutation ability. Under multiple environmental stresses the system could rapidly generate millions of mutants in a few rounds of iterative evolution. Using high-throughput screening, an industrial S. cerevisiae SISc-Δrad52-G4-72 (G4-72) was obtained that is strongly robust and has higher productivity. G4-72 grew stably and produced ethanol efficiently in multiple-stress environments, e.g. high temperature and high osmosis. In a pilot-scale fermentation with G4-72, the fermentation temperature was elevated by 8 °C and ethanol production was increased by 6.9% under the multiple stresses posed by the industrial fermentation substrate. Overall, the GEbCD system presents a powerful tool to rapidly generate abundant mutants and desired hosts, and offers a novel strategy for optimizing microbial chassis with regard to demands posed in industrial applications.

Impact of sequential inoculation timing on the quality of wine fermented by indigenous Lachancea thermotolerans and Saccharomyces cerevisiae

Lack of acidity in wine is one of the main challenges in warm wine-producing regions. The non-Saccharomyces yeast, Lachancea thermotolerans (LT), is well known for its great potential in wine acidification through the production of lactic acid. In this study, an indigenous LT strain was sequentially inoculated with S. cerevisiae (SC). The influence of the timing of SC inoculation on wine quality was extensively studied. Inoculating SC 72 h after the inoculation of LT delayed the peak of fermentation rate, but had no significant influence on the duration of fermentation. The late inoculation (72 h) of SC effectively increased the production of lactic acid, which consequently increased the total wine acidity and decreased the pH. Higher levels of esters were also achieved by the late inoculation. Pilot-scale fermentations were carried out with both Chardonnay and Cabernet Sauvignon to verify the effect of late inoculation. Lactic acid produced in sequential fermentation was 2.73 g/L for Chardonnay wine and 5.35 g/L for Cabernet Sauvignon wine, and the production of ethyl esters was also enhanced. Overall, the results demonstrated that the late inoculation of SC in sequential fermentation with LT improved wine quality by increasing acidity and producing more balanced and aromatic wines.

High-yield porphyrin production through metabolic engineering and biocatalysis.

Porphyrins and their derivatives find extensive applications in medicine, food, energy and materials. In this study, we produced porphyrin compounds by combining Rhodobacter sphaeroides as an efficient cell factory with enzymatic catalysis. Genome-wide CRISPRi-based screening in R. sphaeroides identifies hemN as a target for improved coproporphyrin III (CPIII) production, and exploiting phosphorylation of PrrA further improves the production of bioactive CPIII to 16.5 g L-1 by fed-batch fermentation. Subsequent screening and engineering high-activity metal chelatases and coproheme decarboxylase results in the synthesis of various metalloporphyrins, including heme and the anti-tumor agent zincphyrin. After pilot-scale fermentation (200 L) and setting up the purification process for CPIII (purity >95%), we scaled up the production of heme and zincphyrin through enzymatic catalysis in a 5-L bioreactor, with CPIII achieving respective enzyme conversion rates of 63% and 98% and yielding 10.8 g L-1 and 21.3 g L-1, respectively. Our strategy offers a solution for high-yield bioproduction of heme and other valuable porphyrins with substantial industrial and medical applications.

Statistical optimization and sequential scale-up of α-galactosidase production by Actinoplanes utahensis B1 from shake flask to pilot scale

α-Galactosidase (α-GAL) is a class of hydrolase that releases galactose from galacto-oligosaccharides and synthetic substrates such as pNPG. In this study, the production of α-GAL by Actinoplanes utahensis B1 in submerged fermentation was enhanced by using statistical methods. The effects of temperature, pH, and inoculum percentage on enzyme secretion were optimized using BBD of RSM. The optimized process was scaled up from the shake flask to the laboratory scale (5 L) and to pilot scale (30 L) using KLa based scale-up strategy. By using BBD, a maximum yield of 62.5 U/mL was obtained at a temperature of 28 °C, a pH of 6.9, and an inoculum of 6.4%. Scale-up was performed successfully and achieved a yield of 74.4 U/mL and 76.8 U/mL in laboratory scale and pilot scale fermenters. The TOST was performed to validate the scale-up strategy and the results showed a confidence level of 95% for both scales indicating the perfect execution of scale-up procedure. Through the implementation of BBD and scale-up strategy, the overall enzyme yield has been significantly increased to 76%. This is the first article to explore the scale-up of α-GAL from the A. utahensis B1 strain and provide valuable insights for industrial applications.

Mechanisms for more efficient antibiotics and antibiotic resistance genes removal during industrialized treatment of over 200 tons of tylosin and spectinomycin mycelial dregs by integrated meta-omics

To mitigate the environmental risks posed by the accumulation of antibiotic mycelial dregs (AMDs), this study first attempted over 200 tons of mass production fermentation (MP) using tylosin and spectinomycin mycelial dregs alongside pilot-scale fermentation (PS) for comparison, utilizing the integrated-omics and qPCR approaches. Co-fermentation results showed that both antibiotics were effectively removed in all treatments, with an average removal rate of 92%. Antibiotic resistance gene (ARG)-related metabolic pathways showed that rapid degradation of antibiotics was associated with enzymes that inactivate macrolides and aminoglycosides (e.g., K06979, K07027, K05593). Interestingly, MP fermentations with optimized conditions had more efficient ARGs removal because homogenization permitted faster microbial succession, with more stable removal of antibiotic resistant bacteria and mobile genetic elements. Moreover, Bacillus reached 75% and secreted antioxidant enzymes that might inhibit horizontal gene transfer of ARGs. The findings confirmed the advantages of MP fermentation and provided a scientific basis for other AMDs.

Fermentation of Trichoderma for biological control using local inputs in Costa Rica

Introduction. Supply chain issues have driven up raw material costs and reduced the availability of materials for producing biological control agents. These delays in application could result in increased disease pressure and reduced farm yields. Objective. To determine the effect of different amounts of starch and the use of local ingredients for small and commercial-scale fermentation processes for Trichoderma harzianum. Materials and methods. All trials took place in San José, Costa Rica, between 2016 and 2018. Flask trials were executed to investigate the potential reduction or elimination of starch in commercial fermentation media. Additionally, fermentation vessel trials were conducted to assess the effectiveness of an alternative local medium, encompassing three treatments: 1) Commercial medium as a control, 2) 10% molasses medium, and 3) 10% molasses mediaum with 0.5% yeast extract. Viable spore counts were performed to determine colony forming units (CFU/mL). Results. Reducing starch to 10% of the original medium had no impact on CFU/mL. However, the absence of starch led to uneven growth during fermentation, resulting in solid mycelium accumulations. Molasses medium yielded roughly half the CFU/mL compared to the commercial medium, but it still exceeded the 107 CFU/mL threshold commonly used in studies for biological plant pathogen control. Results from a commercial-scale fermenter mirrored those from pilot-scale fermentation. Conclusion. While reducing starch content in the commercial medium didn't affect growth, the absence of starch caused solid mycelium accumulations, potentially posing issues in commercial production. Employing locally sourced molasses medium on a commercial scale appears feasible while maintaining a viable spore count meeting the minimum field-use specifications. Overall, these findings support the use of these media for Trichoderma production in biological control applications.

Construction of an economical xylose-utilizing Saccharomyces cerevisiae and its ethanol fermentation.

Traditional industrial Saccharomyces cerevisiae could not metabolize xylose due to the lack of a specific enzyme system for the reaction from xylose to xylulose. This study aims to metabolically remould industrial S. cerevisiae for the purpose of utilizing both glucose and xylose with high efficiency. Heterologous gene xylA from Piromyces and homologous genes related to xylose utilization were selected to construct expression cassettes and integrated into genome. The engineered strain was domesticated with industrial material under optimizing conditions subsequently to further improve xylose utilization rates. The resulting S. cerevisiae strain ABX0928-0630 exhibits a rapid growth rate and possesses near 100% xylose utilization efficiency to produce ethanol with industrial material. Pilot-scale fermentation indicated the predominant feature of ABX0928-0630 for industrial application, with ethanol yield of 0.48g/g sugars after 48 hours and volumetric xylose consumption rate of 0.87g/l/h during the first 24 hours. Transcriptome analysis during the modification and domestication process revealed a significant increase in the expression level of pathways associated with sugar metabolism and sugar sensing. Meanwhile, genes related to glycerol lipid metabolism exhibited a pattern of initial increase followed by a subsequent decrease, providing a valuable reference for the construction of efficient xylose-fermenting strains.

Nonconventional yeasts and hybrids for low temperature handcrafted sparkling ciders elaboration in Patagonia

Yeasts play a crucial role in transforming apple must into cider. While Saccharomyces cerevisiae (Sc) has been traditionally associated to cider fermentations worldwide, cryotolerant species such as Saccharomyces uvarum (Su) as well as natural S. cerevisiae × S. uvarum (Sc×Su) hybrids have also been detected in ciders fermented at low temperatures.This study aimed to evaluate the ability of two Patagonian cryotolerant yeast strains (Su and Se) and their interspecific hybrids with a Sc to conduct handcrafted apple must fermentations and a second fermentation process (champenoise method). The main chemical parameters and sensory quality of the resulting sparkling beverages was also analysed.Firstly, Sc×Se and Sc×Su hybrids were evaluated in their fermentative features at laboratory scale. Hybrids were compared with their respective parental species evidencing significant differences in the physicochemical and aromatic composition of the obtained base ciders.Both Su parental strain and the hybrid Sc×Se were selected for performing pilot scale fermentations (250 L) using natural (non-sterilized) apple juice at two different temperatures: 20 °C and 13 °C. Sc parental strain was also evaluated for comparative purposes. All base ciders obtained were then subjected to a second fermentation.A high implantation capacity of both Su and the hybrid was evidenced at the lowest evaluated temperature, while commercial Sc strain was not detected at the final fermentation stage, independently from the temperature. All sparkling ciders exhibited distinct physicochemical profiles. Ciders inoculated with commercial Sc (but effectively fermented with local Sc strains) allowed the development of malolactic fermentation (MLF) in processes carried out at both temperatures. Contrarily, no MLF was observed in ciders inoculated with either Su or the hybrid. Sparkling ciders fermented with Su displayed the highest concentrations of 2-phenylethanol and 2-phenylethyl acetate, regardless of the fermentation temperature. Conversely, ciders fermented with the hybrid at 20 °C exhibited the highest concentrations of ethyl octanoate and ethyl decanoate, contributing to floral and fruity notes in the beverage.Sensory analysis conducted with untrained individuals revealed a preference for sparkling ciders produced with the hybrid strain at both 20 °C and 13 °C. The cider fermented at 20 °C exhibited floral notes, sweetness, and a full body, while ciders fermented at 13 °C displayed moderate acidity and a well-balanced profile. Conversely, a trained panel described the cider fermented at 20 °C with Su as a fruity and acidic beverage, whereas the ciders fermented at 13 °C exhibited intense bitterness and acidity.This study highlights the potential of cryotolerant Saccharomyces species and hybrids in the development of new starter cultures for producing artisanal sparkling ciders with distinctive properties.

Efficient De Novo Biosynthesis of Curcumin in Escherichia coli by Optimizing Pathway Modules and Increasing the Malonyl-CoA Supply.

Curcumin is a natural phenylpropanoid compound with various biological activities and is widely used in food and pharmaceuticals. A de novo curcumin biosynthetic pathway was constructed in Escherichia coli BL21(DE3). Optimization of the curcumin biosynthesis module achieved a curcumin titer of 26.8 ± 0.6 mg/L. Regulating the metabolic fluxes of the β-oxidation pathway and fatty acid elongation cycle and blocking the endogenous malonyl-CoA consumption pathway increased the titer to 113.6 ± 7.1 mg/L. Knockout of endogenous curcumin reductase (curA) and intermediate product detoxification by heterologous expression of the solvent-resistant pump (srpB) increased the titer to 137.5 ± 3.0 mg/L. A 5 L pilot-scale fermentation, using a three-stage pH alternation strategy, increased the titer to 696.2 ± 20.9 mg/L, 178.5-fold higher than the highest curcumin titer from de novo biosynthesis previously reported, thereby laying the foundation for efficient biosynthesis of curcumin and its derivatives.

Reconfiguration of the reductive TCA cycle enables high-level succinic acid production by Yarrowia lipolytica

Succinic acid (SA) is an important C4-dicarboxylic acid. Microbial production of SA at low pH results in low purification costs and hence good overall process economics. However, redox imbalances limited SA biosynthesis from glucose via the reductive tricarboxylic acid (TCA) cycle in yeast. Here, we engineer the strictly aerobic yeast Yarrowia lipolytica for efficient SA production without pH control. Introduction of the reductive TCA cycle into the cytosol of a succinate dehydrogenase-disrupted yeast strain causes arrested cell growth. Although adaptive laboratory evolution restores cell growth, limited NADH supply restricts SA production. Reconfiguration of the reductive SA biosynthesis pathway in the mitochondria through coupling the oxidative and reductive TCA cycle for NADH regeneration results in improved SA production. In pilot-scale fermentation, the engineered strain produces 111.9 g/L SA with a yield of 0.79 g/g glucose within 62 h. This study paves the way for industrial production of biobased SA.

Investigating luxS gene expression in lactobacilli along lab-scale cocoa fermentations

Previous metagenomic analyses have suggested that lactobacilli present potential for Quorum Sensing (QS) in cocoa fermentation, and in the present research, laboratory scale fermentations were carried out to monitor the expression of luxS, a universal marker of QS. For that, 96 h-fermentations were studied, as follows: F0 (non inoculated control), F1 (inoculated with yeasts, lactic acid bacteria, and acetic acid bacteria), F2 (inoculated with yeasts and acetic acid bacteria), F3 (inoculated with yeasts only). The parameters evaluated were: plate counting, quantification of key enzymes and analysis of volatile organic compounds associated with key sensory descriptors, using headspace gas chromatography-mass spectrometry (GC-MS). Furthermore, QS was estimated by the quantification of the expression of luxS genes by Reverse Transcriptase Real-Time PCR. The results demonstrated that microbial succession occurred in pilot scale fermentations, but no statistical differences for microbial enumeration and α-diversity index were observed among experiments and control. Moreover, it was not possible to make conclusive correlations of enzymatic profile and fermenting microbiota, likely due to the intrinsic activity of plant hydrolases. Regarding to the expression of luxS genes, in Lactiplantibacillus plantarum they were active along the fermentation, but for Limosilactobacillus fermentum, luxS was expressed only at early and middle phases. Correlation analysis of luxS expression and production of volatile metabolites evidenced a possible negative association of Lp. Plantarum with fermentation quality. In conclusion, these data corroborate former shotgun metagenomic analysis by demonstrating the expression of luxS by lactobacilli in pilot scale cocoa fermentation and evidence Lp. Plantarum is the main lactic acid bacteria related to its expression.

Pilot-Scale Fermentation of Pseudoalteromonas sp. Strain FDHY-MZ2: An Effective Strategy for Increasing Algicidal Activity.

The role of microorganisms in effectively terminating harmful algal blooms (HABs) is crucial for maintaining environmental stability. Recent studies have placed increased emphasis on bio-agents capable of inhibiting HABs. The bacterium Pseudoalteromonas sp. strain FDHY-MZ2 has exhibited impressive algicidal abilities against Karenia mikimotoi, a notorious global HAB-forming species. To augment this capability, cultures were progressively scaled from shake flask conditions to small-scale (5 L) and pilot-scale (50 L) fermentation. By employing a specifically tailored culture medium (2216E basal medium with 1.5% soluble starch and 0.5% peptone), under precise conditions (66 h, 20 °C, 450 rpm, 30 L/min ventilation, 3% seeding, and constant starch flow), a notable increase in algicidal bacterial biomass was observed; the bacterial dosage required to entirely wipe out K. mikimotoi within a day decreased from 1% to 0.025%. Compared to an unoptimized shake flask group, the optimized fermentation culture caused significant reductions in algal chlorophyll and protein levels (21.85% and 78.3%, respectively). Co-culturing induced increases in algal malondialdehyde and H2O2 by 5.98 and 5.38 times, respectively, leading to further disruption of algal photosynthesis. This study underscores the unexplored potential of systematically utilized microbial agents in mitigating HABs, providing a pathway for their wider application.

High-level production of nervonic acid in the oleaginous yeast Yarrowia lipolytica by systematic metabolic engineering

Nervonic acid benefits the treatment of neurological diseases and the health of brain. In this study, we employed the oleaginous yeast Yarrowia lipolytica to overproduce nervonic acid oil by systematic metabolic engineering. First, the production of nervonic acid was dramatically improved by iterative expression of the genes ecoding β-ketoacyl-CoA synthase CgKCS, fatty acid elongase gELOVL6 and desaturase MaOLE2. Second, the biosynthesis of both nervonic acid and lipids were further enhanced by expression of glycerol-3-phosphate acyltransferases and diacylglycerol acyltransferases from Malania oleifera in endoplasmic reticulum (ER). Third, overexpression of a newly identified ER structure regulator gene YlINO2 led to a 39.3% increase in lipid production. Fourth, disruption of the AMP-activated S/T protein kinase gene SNF1 increased the ratio of nervonic acid to lignoceric acid by 61.6%. Next, pilot-scale fermentation using the strain YLNA9 exhibited a lipid titer of 96.7 g/L and a nervonic acid titer of 17.3 g/L (17.9% of total fatty acids), the highest reported titer to date. Finally, a proof-of-concept purification and separation of nervonic acid were performed and the purity of it reached 98.7%. This study suggested that oleaginous yeasts are attractive hosts for the cost-efficient production of nervonic acid and possibly other very long-chain fatty acids (VLCFAs).

An indigenous bacterial consortium from Argentinean traditional dry sausages as a pilot-scale fermentation starter

An indigenous bacterial consortium from Argentinean traditional dry sausages as a pilot-scale fermentation starter

An end-to-end pipeline for succinic acid production at an industrially relevant scale using Issatchenkia orientalis

Microbial production of succinic acid (SA) at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation for over three decades. Here, we metabolically engineer the acid-tolerant yeast Issatchenkia orientalis for SA production, attaining the highest titers in sugar-based media at low pH (pH 3) in fed-batch fermentations, i.e. 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. We further perform batch fermentation using sugarcane juice medium in a pilot-scale fermenter (300×) and achieve 63.1 g/L of SA, which can be directly crystallized with a yield of 64.0%. Finally, we simulate an end-to-end low-pH SA production pipeline, and techno-economic analysis and life cycle assessment indicate our process is financially viable and can reduce greenhouse gas emissions by 34–90% relative to fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for production of organic acids.

Monitoring the growth dynamics of Tetragenococcus halophilus strains in lupine moromi fermentation using a multiplex-PCR system

ObjectiveThe microbiota of a seasoning sauce fermentation process is usually complex and includes multiple species and even various strains of one species. Moreover, composition and cell numbers of individual strains vary over the course of the entire fermentation. This study demonstrates the applicability of a multiplex PCR system to monitor growth dynamics of Tetragenococcus (T.) halophilus strains in order to evaluate their performance and help to select the most competitive starter strain.ResultsIn a previous study we isolated T. halophilus strains from multiple lupine moromi fermentation processes and characterized them. In this study we wanted to monitor the growth dynamics of these strains in a competitive lupine moromi model fermentation process using a multiplex PCR system. Therefore, pasteurized lupine koji was inoculated with eight different T. halophilus strains, six from lupine moromi, one from an experimental buckwheat moromi fermentation process and the type strain DSM 20,339T, to create the inoculated lupine moromi pilot scale fermentation process. With the multiplex PCR system, we could detect that all strains could grow in lupine moromi but, that TMW 2.2254 and TMW 2.2264 outperformed all other strains. Both strains dominated the fermentation after three weeks with cell counts between 4 × 106 to 4 × 107 CFU/mL for TMW 2.2254 and 1 × 107 to 5 × 107 CFU/mL for TMW 2.2264. The pH dropped to value below 5 within the first 7 days, the selection of these strains might be related to their acid tolerance.

Fractionation of medium-chain-length polyhydroxyalkanoate biosynthesized by pilot-scale production for improving material properties

Medium-chain-length polyhydroxyalkanoate (MCL-PHA), a biodegradable and biocompatible material, is a biopolyester that obtains great attention, especially in the medical area. By focusing on a range of fractions of MCL-PHA, a clear understanding of the thermal behavior and the crystalline structure, especially the tensile properties, have been gained. In this study, we successfully biosynthesized the MCL-PHA in 300 L pilot-scale fermentation by Pseudomonas putida, and all the chemomechanical properties have been characterized. The obtained MCL-PHA (A-original) was fractionated by using a chloroform/methanol mixture into several fractions (A-1, A-2, A-3, and A-4) with different comonomer compositions consisting of 3-hydroxydecanoate (3HD), 3-hydroxyoctanoate (3HO), and 3-hydroxyhexanoate (3HHx). It is crucial to demonstrate the mechanical characteristics of the MCL-PHA fraction in this area of application, which has not previously been studied. We found that the fractions of natural MCL-PHA exhibited distinctive thermal properties with different crystalline patterns, giving rise to the overall behavior of the as-produced MCL-PHA blend primarily dependent on a fraction of high 3HD content. It can be concluded that there are 2 fractions (A-2 and A-3) that have more strength and flexibility than the as-produced MCL-PHA (A-original), indicating that these fractions are suitable for use as medical elastomers.

Effects of native Lachancea thermotolerans combined with Saccharomyces cerevisiae on wine volatile and phenolic profiles in pilot and industrial scale

Wines in the northwest region of China often suffer from poor color stability, low wine acidity, and inelegant aroma description. The Lachancea thermotolerans yeasts with a high yield of lactic acid and pleasant aroma compounds can ameliorate such wines. In this study, the performances of native L. thermotolerans CVE-LT1 co-inoculated with Saccharomyces cerevisiae in different ratios and strategies (simultaneous and sequential mode) were evaluated in pilot scale fermenter (60 L) and industrial scale fermenter (520 hL) of Cabernet Sauvignon grape must with high sugar/low acidity. Results evidenced that the mixed culture of L. thermotolerans and S. cerevisiae can significantly increase lactic acid level (up to 6.98 g/L) and decrease wine pH (up to 3.57). Meanwhile, the color parameters of a* and C* in mixed culture wines were improved. The higher concentrations of volatile compounds were mainly observed in sequential inoculation treatments, including the enhancement of ethyl lactate and 2-phenylethanol. As for phenolic compounds, L. thermotolerans promoted the formation of anthocyanins derivatives and phenolic acids in pilot and industrial fermentation. In conclusion, these results indicated that L. thermotolerans LT1 can be used in hot winery regions to improve the acidity, color indexes, phenolic compounds, and aroma quality of wines.

Pilot Scale Evaluation of Wild Saccharomyces cerevisiae Strains in Aglianico

In winemaking, the influence of Saccharomyces cerevisiae strains on the aromatic components of wine is well recognized on a laboratory scale, but few studies deal with the comparison of numerous strains on a pilot scale fermentation. In this scenario, the present work aimed to validate the fermentative behavior of seven wild S. cerevisiae strains on pilot-scale fermentations to evaluate their impact on the aromatic profiles of the resulting wines. The strains, isolated from grapes of different Italian regional varieties, were tested in pilot-scale fermentation trials performed in the cellar in 1 hL of Aglianico grape must. Then, wines were analyzed for their microbiological cell loads, main chemical parameters of enological interest (ethanol, total sugars, fructose, glucose, total and volatile acidity, malic and lactic acids) and volatile aroma profiles by GC/MS/SPME. Seventy-six volatile compounds belonging to six different classes (esters, alcohols, terpenes, aldehydes, acids, and ketones) were identified. The seven strains showed different trends and significant differences, and for each class of compounds, high-producing and low-producing strains were found. Since the present work was performed at a pilot-scale level, mimicking as much as possible real working conditions, the results obtained can be considered as a validation of the screened S. cerevisiae strains and a strategy to discriminate in real closed conditions strains able to impart desired wine sensory features.