Laboratory-scale Fermentation Research Articles

Optimized production of bacterioruberin from “Haloferax marinum” using one-factor-at-a-time and central composite design approaches

Haloarchaea represents a unique group of microorganisms that have adapted to thrive in high-salt environments. These microbes produce distinctive biomolecules, some of which exhibit extraordinary properties. One such biomolecule is bacterioruberin, a prominent red-pigmented C50 carotenoid commonly found in halophilic archaea, renowned for its antioxidant properties and potential as a functional resource. This study aimed to enhance the culture conditions for optimal production of C50 carotenoids, primarily bacterioruberin, using “Haloferax marinum” MBLA0078. The optimization process involved a combination of one-factor-at-a-time (OFAT) and statistical methodology. Under OFAT-optimized conditions, fed-batch fermentation, and response surface methodology (RSM) optimization, carotenoid production reached 0.954 mg/L, 2.80 mg/L, and 2.16 mg/L, respectively, in a 7-L laboratory-scale fermenter. Notably, RSM-optimized conditions led to a 12-fold increase in productivity (0.72 mg/L/day) compared to the basal DBCM2 medium (0.06 mg/L/day). These findings suggest that strain MBLA0078 holds significant promise for commercial-scale production of bacterioruberin.Graphical

Exploring Putative Kokumi Oligopeptides in Classic Sparkling Wines with a UHPLC-ESI-MS/MS Targeted Protocol.

Some oligopeptides can impart kokumi flavor to foods and beverages, a topic still not addressed in wine. A targeted ultra-high performance liquid-chromatography-mass spectrometry (UHPLC-MS/MS) metabolomics method capable of quantifying both amino acids and oligopeptides in wines was therefore developed and validated, confirming the presence of 50 oligopeptides in wine, most of which had been previously unexplored. In silico screening of the affinity of these oligopeptides to interact with CaSR, the protein necessary to activate kokumi sensations, highlighted 8 dipeptides and 3 tripeptides as putative kokumi compounds. These compounds were ubiquitous in a representative set of Trentodoc classic method sparkling wines, with an average concentration of kokumi oligopeptides of 19.8 mg/L, ranging between 9.1 and 33.3 mg/L. Half of the sparkling wine samples also contained glutamic acid at concentrations equal to or greater than the threshold for the umami taste in wine, namely, 48 mg/L. Sensory tests on the dipeptide Gly-Val confirmed the ability of this novel kokumi compound to significantly modify the perception of complex real wine matrices but not of the simple model one. Preliminary laboratory-scale fermentation tests showed that the oligopeptide profile in wines is linked to the starting grape matrix and that the patterns change by fermenting barley or apple juice with the same yeast.

Impact of Natural Phytosanitary Product Residuals on Yeast Fermentation Performance and Wine Composition.

Although phytosanitary treatments are necessary to protect grapes from parasitic diseases, consumers are increasingly concerned about the use of synthetic phytosanitary products and their possible residues in wine. Pre-harvest phytosanitary treatments are often inevitable, and consequently downtime is required to avoid possible residues on the grapes. Instead, natural phytosanitary products, such as essential oil (EO)-based products, can be applied close to the harvest without specific restrictions, with results that are not only technically convenient but also more attractive for the consumers. Because of the high antimicrobial activity of EO products, in the present study we evaluated the effect of different residual amounts of two new EO-based phytosanitary products on the alcoholic fermentation and the chemical composition of the final fermented products. In particular, two EO-based new formulations, exploitable in organic viticulture management, were evaluated. Increasing concentrations of each formulation were tested during laboratory scale fermentations and in comparison with synthetic and natural commercial phytosanitary products. Growth and fermentation kinetics of a commercial yeast strain of Saccharomyces cerevisiae and the chemical and sensory profiles of the final products were evaluated. Both new formulations showed no significant impact on the growth and fermentation kinetic of S. cerevisiae at any of the concentrations tested. In all trials, alcoholic fermentation was completed in 15 days. Instead, a different chemical composition of the final products was observed. Therefore, these new products might represent an interesting alternative tool to the conventional phytosanitary treatments, being applicable close to the harvest without negative impacts on the kinetics of alcoholic fermentation and also being more acceptable to wine consumers.

Antimicrobial Activity of Chitosan from Different Sources Against Non-Saccharomyces Wine Yeasts as a Tool for Producing Low-Sulphite Wine

Chitosan is used as an antimicrobial agent in different agri-food applications; in winemaking, the use of chitosan from Aspergillus niger is authorized, but other sources of chitin, and consequently of chitosan, are available, such as crustaceans and insects. This work investigates the antimicrobial efficiency of chitosan from crustaceans and insects (Hermetia illucens) against non-Saccharomyces yeasts in wine. For this aim, the first step was to evaluate the effect of crustacean chitosan, tested both alone and in combination with low sulphur dioxide (SO2) concentrations, on the cell viability of 20 non-Saccharomyces strains in the first step of fermentations inoculated with each strain. Furthermore, the strain resistance to crustacean- and insect-based chitosan was evaluated in agarized media, together with the addition of different antimicrobial concentrations. Finally, the efficiency of different antimicrobial treatments was evaluated during laboratory-scale fermentations inoculated with a selected S. cerevisiae strain. The tested strains exhibited medium/high resistance to the chitosan; in some cases, the behaviour varied in the function of species/strain, and only four strains exhibited different resistance levels, depending on the chitosan source. The addition of chitosan alone during fermentation inoculated with S. cerevisiae showed lower antimicrobial activity than SO2, but the combined use with SO2 showed a better effect than chitosan alone. The evaluation of the suitability of chitosan obtained from a sustainable source, such as insects, will allow us to give new information on the future applications of this natural compound for the production of wine with low sulphite content.

Robust prediction for characteristics of digestion products in an industrial-scale biogas project via typical non-time series and time-series machine learning algorithms

Anaerobic digestion (AD) is a well-established pathway for treating agricultural organic waste, and machine learning has emerged as a novel tool to predict its product performance. In prior research, the majority of studies concentrated on non-time series models for laboratory-scale fermentation data. Consequently, the generalization performance of these models was significantly constrained, particularly in the context of industrial-scale biogas projects. Thus, in this study, typical non-time series models (GBR and RF) and time-series models (LSTM, CNN-LSTM, and DA-LSTM) after hyperparameter optimization were chosen to accurately predict the characteristics of digestion products in a biogas project. The ideal GBR model for CH4 content was obtained, and the R2 values of the test set and training set were 0.93 (RMSE=1.11) and 0.97 (RMSE=0.69), respectively. Temperature was the most important parameter for biogas production according to feature importance and SHAP analysis of the RF model. The DA-LSTM was superior to LSTM and CNN-LSTM for the prediction of biogas production, and the R2 of DA-LSTM was 0.87 (RMSE=1048.14) with a seq_len of 10 d. This study provides direction for high-efficiency biogas production in wet biogas projects with the aid of reliable machine learning models.

Cell Penetrating Peptide Enhances the Aphidicidal Activity of Spider Venom-Derived Neurotoxin.

HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 μg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 μg/mL and 465.2 μg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 μg/mL-over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications.

Refermentation with yeast and lactic acid bacteria isolates: a strategy to improve the flavor of green coffee beans.

Yeast and lactic acid bacteria (LAB) play an important part in the post-harvest fermentation of coffee. This study applied lab-scale fermentation to commercial green coffee beans using dry coffee pulp as the substrate, with the aim of modifying coffee-bean flavor. In addition to spontaneous fermentation, yeast and LAB isolated from coffee beans and dried coffee pulp were added during fermentation. Co-inoculation of yeast and LAB showed a significant effect on the chlorogenic acid content after between 24 and 72 h of fermentation. Acetic, citric, malic, lactic, and quinic acids were shown to be affected significantly (P < 0.05) by fermentation and inoculation. Gas chromatography detected that esters, alcohols, aldehydes, furans, and pyrazines were the primary compounds in the coffee beans. Certain volatile groups were present in greater concentrations and broader varieties within the inoculated beans. The highest cupping scores were given to beans that had been co-inoculated with yeast and LAB. Overall, the use of yeasts and LAB starters showed potential to create coffee beverages with desirable characteristics by standardized fermentation. © 2024 Society of Chemical Industry.

FERMENTATION, PURIFICATION AND IN-VITRO EVALUATION OF ANTIMICROBIAL, ANTITUMOR AND ANTIOXIDANT COMPOUNDS FROM A HIMALAYAN ACTINOBACTERIA STRAIN STREPTOMYCES SP. PU-AK14

This study reports the laboratory scale fermentation, compounds purification and bioactivity screening of the strain Streptomyces sp. PU-AK14 isolated from Himalayan mountains range, Pakistan, for its potential to produce antimicrobial, antioxidant and antitumor compounds. For this purpose, laboratory fermentation (20 L working volume) was performed followed by extraction of the compounds by XAD adsorbent gel using the solvents methanol and ethyl acetate. Further purification of the compounds was done by silica gel column chromatography, sephadex column and preparative TLC. A total of 18 partially purified compounds were retrieved from crude extract and were screened for biological activity. The crude extract was more active against gram negative bacteria with MIC of 0.1 mg/ml against Klebsiella pneumoniae and Escherichia coli. Eight of the partially purified compounds exhibited activity against MRSA whereas five fractions were active against gram negative bacteria. Seven partially purified fractions exhibited promising antioxidant activity with radical scavenging activity ranging from 78.2 to 42.34 percent. Cytotoxicity of partially purified compounds ranged from 75% to 100% larval mortality at 100mg/ml against Artemia salina and 50% to 52% cell mortality against HCT 116 colorectal cancer cell line at 100mg/ml. The best anti-tumor activity was exhibited by the fraction AK141111L. The study suggested that Streptomyces sp. PU-AK14 is a potential source of chemotherapeutically useful compounds.

Engineering Implementation of the Acosta Fermentation Method to Obtain Cuban Schnapps with Reduced Concentrations of Higher Alcohols

The Acosta method involves rewiring the yeast metabolic pathway to enhance biomass production and prevent a significant increase in higher alcohols during the late stages of fermentation. This study aimed to assess fermentation conditions to achieve Cuban schnapps with reduced concentrations of higher alcohols and replicate the process on an industrial scale. To achieve this, the quality of final sugarcane molasses for fermentation by Saccharomyces cerevisiae (S. cerevisiae) yeast was evaluated. Industrial pre-fermentation and fermentation processes were successfully conducted, followed by laboratory-scale fermentation using the Acosta fermentation method to determine crucial parameters for industrial implementation. Operational parameters for fermentation were identified from the following results: 13.5 °Brix seeding, metabolic pathway inversion of S. cerevisiae at 16 h, and an air concentration of 0.1 m3/min. The resulting Cuban schnapps obtained using this method exhibited a concentration of higher alcohols of 132.5 mg/L, a value that is within the standard parameters, showing a positive impact of this fermentation method on the quality of the schnapps. Scaling up this method to an industrial level, in addition to offering higher quality products and being an economically viable alternative, also stands out for its sustainable and environmentally friendly aspect, and results in higher production of yeast biomass as a byproduct, which can be used for various purposes, such as animal feed. This method constitutes an important update to the schnapps production process as a technological improvement that respects sustainable production trends and the characteristics of the final product.

New Method of Obtaining Proteolytic Enzymatic Preparation from Fungal Strain Fusarium gibbosum CNMN FD 12

Proteases, that are able to hydrolyze the peptide bonds in proteins and polypeptides, occupy a key position in diverse fields, such as food, agriculture, animal feed. Considering the increasing demand for microbial proteases, identification of new way for improving the productivity of producers is of great importance. The aim of the present study was to evaluate the effect of a new coordination compound [Fe(H2L)(H2O)2](NO3)3∙2.5H2O (where H2L = 2,6-diacetylpyridine bis(picolinoylhydrazone)) on the activity of proteolytic complex synthesized by the micromycete Fusarium gibbosum CNMN FD 12 under the submerged fermentations in shake flask, as well as in lab-scale fermenter. It was found that [Fe(H2L)(H2O)2](NO3)3∙2.5H2O exerts a significant positive effect on proteolytic activity in F. gibbosum CNMN FD 12 both in flasks and bioreactor and can be used as stimulator and regulator of proteases activity. By varying the concentration of the coordination compound and the duration of cultivation, it is possible to obtain proteolytic complexes enriched in different types of proteases (acid, neutral or alkaline), depending on the practical needs.

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.

Evaluation of stress tolerance and design of alternative culture media for the production of fermentation starter cultures in cacao

Ecuador is one of the world's leading producers of cacao beans, and Nacional x Trinitario cacao represents one of the most distinctive varieties due to its flavor and aroma characteristics. This study aimed to evaluate the effect of the starter culture isolated from microbial diversity during the spontaneous fermentation of Nacional x Trinitario cacao. A total of 249 microbial isolates were obtained from spontaneous culture, with Lactiplantibacillus (45 %), Saccharomyces (17 %), and Acetobacter (2 %) being the most relevant genera for fermentation. Tolerance tests were conducted to select microorganisms for the starter culture. Lactiplantibacillus plantarum exhibited the highest tolerance at pH 5 and 6 % ethanol and tolerated concentrations up to 15 % for glucose and fructose. Acetobacter pasteurianus grew at pH 2 and 6 % ethanol, tolerating high sugar concentrations of up to 15 % for glucose and 30 % for fructose, with growth observed in concentrations up to 5 % for lactic and acetic acid. Subsequently, a laboratory-scale fermentation was conducted with the formulated starter culture (SC) comprising S. cerevisiae, L. plantarum, and A. pasteurianus, which exhibited high tolerance to various stress conditions. The fermentation increased alcoholic compounds, including citrusy, fruity aromas, and floral notes such as 2-heptanol and phenylethyl alcohol, respectively 1.6-fold and 5.6-fold compared to the control. Moreover, the abundance of ketones 2-heptanone and 2-nonanone increased significantly, providing sweet green herbs and fruity woody aromas. Cacao fermented with this SC significantly enhanced the favorable aroma-producing metabolites characteristic of Fine-aroma cacao. These findings underscore the potential of tailored fermentation strategies to improve cacao product quality and sensory attributes, emphasizing the importance of ongoing research in optimizing fermentation processes for the cacao industry.

Circular economyeast: Saccharomyces cerevisiae as a sustainable source of glucans and its safety for skincare application

Glucans, a polysaccharide naturally present in the yeast cell wall that can be obtained from side streams generated during the fermentation process, have gained increasing attention for their potential as a skin ingredient. Therefore, this study focused on the extraction method to isolate and purify water-insoluble glucans from two different Saccharomyces cerevisiae strains: an engineered strain obtained from spent yeast in an industrial fermentation process and a wild strain produced through lab-scale fermentation. Two water-insoluble extracts with a high glucose content (> 90 %) were achieved and further subjected to a chemical modification using carboxymethylation to improve their water solubility. All the glucans' extracts, water-insoluble and carboxymethylated, were structurally and chemically characterized, showing almost no differences between both yeast-type strains. To ensure their safety for skin application, a broad safety assessment was undertaken, and no cytotoxic effect, immunomodulatory capacity (IL-6 and IL-8 regulation), genotoxicity, skin sensitization, and impact on the skin microbiota were observed. These findings highlight the potential of glucans derived from spent yeast as a sustainable and safe ingredient for cosmetic and skincare formulations, contributing to the sustainability and circular economy.

Bringing a scalable adaptive hybrid modeling framework closer to industrial use: Application on a multiscale fungal fermentation.

Digitalization has paved the way for new paradigms such as digital shadows and digital twins for fermentation processes, opening the door for real-time process monitoring, control, and optimization. With a digital shadow, real-time model adaptation to accommodate complex metabolic phenomena such as metabolic shifts of a process can be monitored. Despite the many benefits of digitalization, the potential has not been fully reached in the industry. This study investigates the development of a digital shadow for a very complex fungal fermentation process in terms of microbial physiology and fermentation operation on pilot-scale at Novonesis and the challenges thereof. The process has historically been difficult to optimize and control due to a lack of offline measurements and an absence of biomass measurements. Pilot-scale and lab-scale fermentations were conducted for model development and validation. With all available pilot-scale data, a data-driven soft sensor was developed to estimate the main substrate concentration (glucose) with anormalized root mean squared error(N-RMSE)of 2%. This robust data-driven soft sensor was able to estimate accurately in lab-scale (volume < 20× pilot) with a N-RMSE of 7.8%. A hybrid soft sensor was developed by combining the data-driven soft sensor with a mass balance to estimate the glycerol and biomass concentrations on pilot-scale data with N-RMSEs of 11% and 21%, respectively. A digital shadow modeling framework was developed by coupling a mechanistic model (MM) with the hybrid soft sensor. The digital shadow modeling framework significantly improved the predictability compared with the MM. The contribution of this study brings the application of digital shadows closer to industrial implementation. It demonstrates the high potential of using this type of modeling framework for scale-up and leads the way to a new generation of in silico-based process development.

Modeling and simulation of an integrated bioethanol-hydrogen production process

Hydrogen (H2) production from renewable feedstocks such as bioethanol represents one of the sustainable and environmentally benign methods of making the proposed utilization of H2 as energy carrier of the future a reality. However, successful implementation of this technology depends on several factors which include understanding of the technicality, as well as process design and modelling. Generally, this area of research has not received adequate attentions. In this article, an integrated process for producing H2 through starch hydrolysis, fermentation, and ethanol steam reforming (ESTR) process has been proposed. Enzymatic hydrolysis of starch was performed to obtain the fermentable sugar (hydrolysate). In addition, batch fermentation experiments were carried out using a laboratory scale fermenter with yeast (Saccharomyces cerevisiae) culture adapted for anaerobic conversion of the hydrolysate to bioethanol. Subsequently, kinetic data obtained from these experiments was employed to guide thermodynamic modeling of fermentation process which was coupled with the ESTR model and was implemented in the Aspen HYSYS simulator. The results ultimately revealed pertinent information which could find applications in the implementation of the proposed integrated process for bioethanol and H2 production.

Optimization of bacterioruberin production from Halorubrum ruber and assessment of its antioxidant potential

Haloarchaea produce bacterioruberin, a major C50 carotenoid with antioxidant properties that allow for its potential application in the food, cosmetic, and pharmaceutical industries. This study aimed to optimize culture conditions for total carotenoid, predominantly comprising bacterioruberin, production using Halorubrum ruber MBLA0099. A one-factor-at-a-time and statistically-based experimental design were applied to optimize the culture conditions. Culture in the optimized medium caused an increase in total carotenoid production from 0.496 to 1.966 mg L− 1 Maximal carotenoid productivity was achieved in a 7-L laboratory-scale fermentation and represented a 6.05-fold increase (0.492 mg L–1 d–1). The carotenoid extracts from strain MBLA0099 exhibited a 1.8–10.3-fold higher antioxidant activity in vitro, and allowed for a higher survival rate of Caenorhabditis elegans under oxidative stress conditions. These results demonstrated that Hrr. ruber MBLA0099 has significant potential as a haloarchaon for the commercial production of bacterioruberin.

Developing a novel selection method for alcoholic fermentation starters by exploring wine yeast microbiota from Greece.

The selection of native yeast for alcoholic fermentation in wine focuses on ensuring the success of the process and promoting the quality of the final product. The purpose of this study was firstly to create a large collection of new yeast isolates and categorize them based on their oenological potential. Additionally, the geographical distribution of the most dominant species, Saccharomyces cerevisiae, was further explored. Towards this direction, fourteen spontaneously fermented wines from different regions of Greece were collected for yeast typing. The yeast isolates were subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M13 was used, combined with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. All yeast isolates were scrutinized for their sensitivity to killer toxin, production of non-desirable metabolites such as acetic acid and H2S, β-glucosidase production and resistance to the antimicrobial agent; SO2. In parallel, S. cerevisiae isolates were typed at strain level by interdelta - PCR genomic fingerprinting. S. cerevisiae strains were examined for their fermentative capacity in laboratory scale fermentation on pasteurized grape must. Glucose and fructose consumption was monitored daily and at the final point a free sorting task was conducted to categorize the samples according to their organoleptic profile. According to our results, among the 190 isolates, S. cerevisiae was the most dominant species while some less common non-Saccharomyces species such as Trigonopsis californica, Priceomyces carsonii, Zygosaccharomyces bailii, Brettanomyces bruxellensis and Pichia manshurica were identified in minor abundancies. According to phenotypic typing, most isolates were neutral to killer toxin test and exhibited low acetic acid production. Hierarchical Cluster Analysis revealed the presence of four yeast groups based on phenotypic fingerprinting. Strain level typing reported 20 different S. cerevisiae strains from which 65% indicated fermentative capacity and led to dry wines. Sensory evaluation results clearly discriminated the produced wines and consequently, the proposed yeast categorization was confirmed. A novel approach that employs biostatistical tools for a rapid screening and classification of indigenous wine yeasts with oenological potential, allowing a more efficient preliminary selection or rejection of isolates is proposed.

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.

Maximizing Bioethanol Production from Eucalyptus globulus Using Steam Explosion Pretreatment: A Multifactorial Design and Fermenter Development for High Solid Loads

Steam explosion pretreatment is suitable for bioethanol production from Eucalyptus globulus wood. Multifactorial experiment designs were used to find the optimal temperature and residence time required to obtain the best glucose yield from the enzymatic hydrolysis of pretreated materials. The chemical composition, crystallinity index, morphology and polymerization degree of the pretreated materials were correlated with enzymatic accessibility. Simultaneous saccharification and fermentation (SSF) using a fed-batch strategy was applied to three different laboratory-scale fermenters. The optimization of the pretreatment was obtained at 208 °C and 11 min. However, the enzymatic hydrolysis performance did not show significant differences from the material obtained at 196 °C and 9.5 min, which was determined to be the real optimum, owing to its lower energy requirement. The vertical fermenter with type “G” blades and the horizontal fermenter with helical blades were both highly efficient for reaching ethanol yields close to 90% based on dry wood, and ethanol concentrations close to 9.0% v/v.

Development of an Ultrasound-Assisted Emulsification Microextraction Method for the Determination of Volatile Compounds in Wines

A fast and simple method based on ultrasound-assisted emulsification microextraction (USAEME) was developed for the analysis of volatile compounds in wines. A full factorial 24 screening design was built to investigate the main factors affecting the extraction of volatile components, namely the volume of extraction solvent, sonication time, salt content, and pH. Then, the factors with significant effects were optimized using an I-optimal design. The optimal value for all the variables studied was reached under the following experimental conditions: volume of extraction solvent 200 μL and salt content 5% m/v. The suitability of the optimized method was evaluated, resulting in very good linearity with coefficients of determination (R2) higher than 0.995 in all cases, while repeatability was lower than 8.4% except for d-limonene and p-cymene. Recoveries higher than 82% were observed for the groups of ethyl esters, acetate esters, alcohols, and terpenoid alcohols (linalool, α-terpineol). The recovery of acids ranged from 70.5% to 88.9%, whereas the three monoterpenes studied (d-limonene, γ-terpinene, p-cymene) were not extracted satisfactorily. The proposed method was effectively applied for the analysis of volatile compounds in laboratory-scale fermentations with selected strains of Saccharomyces cerevisiae.