Industrial-scale Fermentation Research Articles

ORP-regulated natural accumulation of pyruvate in Actinobacillus succinogenes 130Z

Pyruvate, a pivotal metabolite in the glycolytic pathway, typically confronts substantial barriers to its natural accumulation within microbial cells. This study successfully facilitated the natural accumulation of pyruvate in Actinobacillus succinogenes 130Z by fine-tuning the oxidation-reduction potential (ORP) in the fermentation milieu. A mechanistic exploration revealed that the accumulation of pyruvate was optimized when ORP conditions favorably modulated pyruvate kinase activity and concurrently suppressed succinate dehydrogenase activity. By integrating the influence of metal ions on enzymatic functions with an innovative aluminum ion-mediated ORP control strategy, we achieved a pyruvate yield of 27.54 g/L over 20 hours, which constitutes an 89.54 % increase compared to the baseline. Additionally, the production rate of pyruvate reached 1.38 g/L·h. This investigation not only elucidates the metabolic underpinnings that facilitate the natural enrichment of glycolytic intermediates in Actinobacillus succinogenes 130Z but also lays a robust theoretical foundation for the industrial-scale fermentation of pyruvate. Moreover, the capability to efficiently and rapidly concentrate essential platform metabolites within the glycolytic pathway is of paramount significance, potentially propelling forward the research and synthesis of various downstream metabolic products.

Omics-based analysis of Akkermansia muciniphila cultivation in food-grade media

Background and Aim: Over the past years, the gut microbiota and its correlation to health and disease has been studied extensively. In terms of beneficial microbes, an increased interest in Akkermansia muciniphila (A. muciniphila ) has been observed since its discovery. Direct evidence for the role of A. muciniphila in host health has been provided in both mice and human studies. However, for human interventions with A. muciniphila cells, industrial-scale fermentations are needed, and hence, the used cultivation media should be free of animal-derived components, food-grade, non-allergenic and allow for efficient growth to high densities to provide cost-effective production platforms. In this study, we assessed the growth and performance of A. muciniphila in batch bioreactors using newly developed plant-based media. Methods: The bioreactors were supplemented with varying carbon sources, including different ratios of N-acetylglucosamine (GlcNAc) and glucose. We monitored the growth of A. muciniphila in the plant-based medium using optical density (OD600) measurements and microscopy. In addition, we used a combination of biochemical analysis as well as transcriptional and proteomics analysis to gain detailed insight into the physiology. Results: Comparisons between growth on these media and that on mucin revealed differences at both transcriptome and proteome levels, including differences in the expression of glycosyltransferases, signaling proteins, and stress response. Furthermore, elongated cells and higher OD600 values were observed using the plant-based media as compared to cultivation media containing mucin. Conclusion: These differences do not hamper growth, and therefore, our data suggest that the food-grade medium composition described here could be used to produce A. muciniphila with high yields for therapeutic purposes.

Decarboxylative Ketonization of Aliphatic Carboxylic Acids in a Continuous Flow Reactor Catalysed by Manganese Oxide on Silica.

The sustainable synthesis of long carbon chain molecules from carbon dioxide, water and electricity relies on the development of waste-free, highly selective C-C bond forming reactions. An example for such a power-to-chemicals process is the industrial-scale fermentation for the production of hexanoic acid. Herein, we describe how this product is transformed into 6-undecanone via decarboxylative ketonization using a heterogeneous manganese oxide/silica catalyst. The reaction reaches full conversion with near-complete selectivity when carried out in a continuous flow reactor, requires no solvent or carrier gas, and releases carbon dioxide and water as the only by-products. The reactor was operated for several weeks with no loss of reactivity, producing 7 kg of 6-undecanone from 10 g of catalyst and achieving a productivity of 1.135 kg per litre of reactor volume per hour. 6-Undecanone and other long-chain ketones accessible this way can be hydrogenated to industrially meaningful alkanes, or converted into valuable fatty acids via a hydrogenation/elimination/isomerizing hydrocarboxylation sequence.

Dynamic changes in metabolomics and flavoromics during industrial scale fermentation of Chinese traditional soy sauce

Flavor substances are crucial to the quality of soy sauce and closely related to the intermediate metabolites. This study systematically explored the dynamic changes in physicochemical characteristics, metabolomics and flavoromics during traditional Chinese soy sauce fermentation. The results indicated that the amino acid nitrogen gradually increased to 0.95 g/100 mL on the 13th day and then stabilized, and the changes of reducing sugars increased to 66.40 mg/mL on the 7th day and then slowly decreased. A total of 90 volatile compounds and 1172 non-volatile metabolites were detected. In the early fermentation period (1–7 day), alcohols, aldehydes and ketones were the main volatile compounds, but as fermentation proceeded, phenols and esters gradually accumulated. The total content of volatile compounds, mainly alcohols and phenols, reached the highest value on the 23rd day. Non-volatile metabolites were mainly amino acids, organic acids, lipids and carbohydrates, and the content and types of varied with fermentation time. KEGG analysis indicates there were significant changes in the proline and arginine metabolism in the middle and late periods (7–28 day) compared to the early fermentation period. Correlation analysis indicating a delicate balance between the transformation of metabolites and the formation of volatile compounds in traditional Chinese soy sauce.

Effects of Phenolic Evolution on Color Characteristics of Single-Cultivar Vitis vinifera L. Marselan and Merlot Wines during Vinification and Aging

The loss of red hue in dry red wine has been a persistent issue for wine enterprises in western China. We investigated the changes in anthocyanins and non-anthocyanin phenols during the industrial-scale fermentation and one-year bottle aging of Vitis vinifera L. Merlot and Vitis vinifera L. Marselan, respectively, using the grapes in the Ningxia region. We also examined their correlation with color characterization. The study found that both anthocyanins and non-anthocyanin phenolics were rapidly extracted from grapes during alcohol fermentation. However, their concentrations decreased rapidly during malolactic fermentation. On the other hand, Vitisin A and Vitisin B were formed during alcoholic fermentation and decreased slowly from malolactic fermentation to storage period. Directly polymerized pigments (F-A and A-F), bridged polymerized pigments (A-e-F), and flavanyl-pyranoanthocyanins (A-v-F) from the reactions of anthocyanins (A) and flavan-3-ols (F), as well as pinotins were generated during the later stages of alcoholic fermentation, and remained at a high level throughout malolactic fermentation and bottle storage. Partial least squares regression and Pearson correlation analyses revealed that the red hue (a* value) of ‘Merlot’ and ‘Marselan’ wines was closely associated with monomeric anthocyanins and F-A type pigments. Furthermore, four pinotin components were positively correlated with the red hue (a* value) of ‘Merlot’ wine. These primary red components of the two varieties had a positive correlation with the level of flavan-3-ols. The data suggest that elevating the flavan-3-ol concentration during fermentation aids in improving the color stability of red wine.

The evolution of anaerobic growth in Saccharomycotina yeasts.

Humans rely on the ability of budding yeasts to grow without oxygen in industrial scale fermentations that produce beverages, foods, and biofuels. Oxygen is deeply woven into the energy metabolism and biosynthetic capabilities of budding yeasts. While diverse ecological habitats may provide wide varieties of different carbon and nitrogen sources for yeasts to utilize, there is no direct substitute for molecular oxygen, only a range of availability. Understanding how a small subset of budding yeasts evolved the ability to grow without oxygen could expand the set of useful species in industrial scale fermentations as well as provide insight into the cryptic field of yeast ecology. However, we still do not yet appreciate the full breadth of species that can growth without oxygen, what genes underlie this adaptation, and how these genes have evolved.

Determining the linear correlation between dielectric spectroscopy and viable biomass concentration in filamentous fungal fermentations

ObjectivesDielectric spectroscopy is commonly used for online monitoring of biomass growth. It is however not utilized for biomass concentration measurements due to poor correlation with Cell Dry Weight (CDW). A calibration methodology is developed that can directly measure viable biomass concentration in a commercial filamentous process using dielectric values, without recourse to independent and challenging viability determinations.ResultsThe methodology is applied to samples from the industrial scale fermentation of a filamentous fungus, Acremonium fusidioides. By mixing fresh and heat-killed samples, linear responses were verified and sample viability could be fitted with the dielectric Delta C values and total solids concentration. The study included a total of 26 samples across 21 different cultivations, with a legacy at-line viable cell analyzer requiring 2 ml samples, and a modern on-line probe operated at-line with 2 different sample presentation volumes, one compatible with the legacy analyzer, a larger sample volume of 100 ml being compatible with calibration for on-line operation. The linear model provided an {R}^{2} value of 0.99 between Delta C and viable biomass across the sample set using either instrument. The difference in ∆C when analyzing 100 mL and 2 mL samples with an in-line probe can be adjusted by a scalar factor of 1.33 within the microbial system used in this study, preserving the linear relation with {R}^{2} of 0.97.ConclusionsIt is possible to directly estimate viable biomass concentrations utilizing dielectric spectroscopy without recourse to extensive and difficult to execute independent viability studies. The same method can be applied to calibrate different instruments to measure viable biomass concentration. Small sample volumes are appropriate as long as the sample volumes are kept consistent.

Production of gamma-aminobutyric acid (GABA) by Bacillus subtilis BBEL02 fermentation using nitrogen-rich industrial wastes as crude feedstocks

Industrial-scale fermentation of γ-aminobutyric acid (GABA) is often limited by high cost of the starting raw materials. Increasing commercial applications of GABA in various industries have urged the research on finding the alternative substrates for the GABA production. The effects of concentration of different types of carbon and nitrogen sources; concentration of monosodium glutamate (MSG) in the defined medium for GABA production using the newly isolated Bacillus subtilis BBEL02 were studied. The feasibility of corn steep liquor (CSL) and soybean hydrolysate (SBH) as the replacement of nitrogen sources in the fermentation media for GABA production was also investigated. Highest GABA content (10.9 gL−1) and productivity (3.7 gL−1d−1) were achieved with defined medium containing 1 % (w/v) glucose, 2 % (v/v) SBH and 5 % (w/v) MSG. Dissolved oxygen (DO) of 80 % in 2-L fermenter presented the highest productivity (4.2 gL−1d−1) and GABA concentration (12.5 gL−1), however DO of 50 % was preferred because of the lower rotation speed to prevent cells deterioration.

Seasonal dynamics of microbiota and physicochemical indices in the industrial-scale fermentation of Sichuan Baoning vinegar.

We investigated the impact of seasons of the year on microbiota and physicochemical indices in industrial-scale fermentation of Sichuan Baoning vinegar. Illumina HiSeq sequencing results showed significant differences (P<0.05) between the microbiomes of vinegar Pei in every-two seasons, except for bacterial communities between summer and autumn. Total acid, reducing sugar, starch, and alcohol contents of vinegar Pei from the same sampling day of each season were measurably different. Although total acid content in vinegar Pei was similar at the end of fermentation (P>0.05), the increase in total acidity was highest in the autumn. Acetic acid content in raw vinegar was highest in the autumn (3472.42mg/100mL), and lowest in the summer (2304.01mg/100mL). This study provides a theoretical basis for the production of Sichuan bran vinegar with consistent quality and provides insights into the quality control of traditional fermented foods.

Food processing by-products as sources of hydrophilic carbon and nitrogen for sophorolipid production

Valorization of unavoidable food supply chain waste via biotechnology would be beneficial to the implementation of circular economics and may help increase market competitiveness of the final product via reduced feedstock costs. The suitability of post-milling wheat feed, potato processing scraps and sugar-beet pulps to act as the sole source of hydrophilic carbon (glucose), nitrogen and micronutrients for sophorolipid production using Starmerella bombicola was assessed. While all three feedstocks were viable for sophorolipid production, potato scraps were the most promising feedstock, with product titers of 60.5 and 77.8 g/L from batch and fed-batch bioreactor fermentations, respectively. The viability of potato hydrolysate as a feeding media was also demonstrated, with a three-fold increase in productivity of fed-batch over batch fermentation. This bodes well for future use of potato hydrolysate in more sophisticated fermentation schemes involving continuous feeding and in-situ separation. To holistically evaluate the viability of the potato waste as fermentation feedstock, supply chain modeling was conducted using the UK as a case study. The modeling identified ideal locations for industrial scale fermentation of the potato waste, such as North-West England, East Midlands and Central Scotland. The scale of the facility was shown to greatly alter the complexity of the supply chain, leading to significant changes in environmental and fiscal costs. Practical processing considerations and the implication on process techno-economics are also discussed. The insights provided will be useful for future development of sustainable feedstock for industrial sophorolipid production and industrial biotechnology in general.

The ability of Pichia kudriavzevii to tolerate multiple stresses makes it promising for developing improved bioethanol production processes.

Thermotolerant ethanol fermenting yeasts have been extensively used in industrial bioethanol production. However, little is known about yeast physiology under stress during bioethanol processing. This study investigated the physiological characteristics of the thermotolerant yeast Pichia kudriavzevii, strains NUNS-4, NUNS-5 and NUNS-6, under the multiple stresses of heat, ethanol and sodium chloride. Results showed that NUNS-4, NUNS-5 and NUNS-6 displayed higher growth rates under each stress condition than the reference strain, Saccharomyces cerevisiae TISTR5606. Maximum specific growth rates under stresses of heat (45°C), 15% v/v ethanol and 1·0 M sodium chloride were 0·23 ± 0·04 (NUNS-4), 0·11 ± 0·01 (NUNS-5) and 0·15 ± 0·01 h-1 (NUNS-5), respectively. Morphological features of all yeast studied changed distinctly with the production of granules and vacuoles when exposed to ethanol, and cells were elongated under increased sodium chloride concentration. This study suggests that the three P. kudriavzevii strains are potential candidates to use in industrial-scale fermentation due to a high specific growth rate under multiple stress conditions. Multiple stress-tolerant P. kudriavzevii NUNS strains have received much attention not only for improving large-scale fuel ethanol production, but also for utilizing these strains in other biotechnological industries.

Native Non-Saccharomyces Yeasts as a Tool to Produce Distinctive and Diverse Tamjanika Grape Wines.

The enological potential of two previously characterized indigenous yeast isolates, Hanseniaspora uvarum S-2 and Candida famata WB-1, in pure and sequential inoculation with commercial yeast Saccharomyces cerevisiae QA23 were analyzed in industrial-scale vinification of the grape variety Tamjanika. Their contribution to the quality and aroma profile was investigated by quantifying volatile compounds and wine sensory evaluation. Both yeast isolates were able to complete alcoholic fermentation, to reduce ethanol concentration up to 1.06% v/v (in monoculture) in comparation to S. cerevisiae QA23, and to enhance aroma and sensory profile. Based on calculated odor activity values (OAV), p-cymene, ethyl hexanoate, ethyl octanoate, and ethyl decanoate were the major aroma volatile compounds in all Tamjanika wine samples. Analyzed yeast strains significantly affected relative contribution of volatile compounds and can be considered responsible for the differences and uniqueness of the obtained wine samples. Besides confirmation of good enological and fermentative characteristics, selected isolates can be characterized as high ester-producing strains with potential to enhance the floral and fruity aromas of wine. The present study represents a further step toward the use of indigenous yeast isolates at industrial-scale fermentation in order to ensure the regional signature of Tamjanika wine.

Real-time monitoring of kefir-facilitated milk fermentation using microbial potentiometric sensors

Considering that fermentation is a microorganism-mediated redox process, an underlying hypothesis was postulated that the microbial potentiometric sensor technology could be used to monitor dairy fermentation or other similar food production processes. It was also hypothesized that the signal characteristics could be exploited to correlate the mass of inoculum and the fermentation completion time. Three objectives were addressed to test these hypotheses. First, a set of fermentation experiments were employed to measure the changes in the open-circuit potential signals from two carbon-based microbial potentiometric sensor (MPS) electrodes exposed to varying kefir inoculum to milk ratios. Second, the MPS signal patterns were analyzed to determine what signals were indicative of completion of the fermentation process. Third, regression analyses were conducted to determine the level of correlation between the fermentation completion time and the mass of the inoculum under constant conditions. The hypothesized capabilities of this technology for monitoring kefir fermentation processes of milk were validated. The MPS technology could be used to monitor kefir fermentation in real-time with high reproducibility. The regression analysis approach was able to discern a correlation between the fermentation completion time and the mass of kefir inoculum characterized with coefficients of determination R2 > 0.94. Furthermore, the results from the MPS signal patterns offered unique perspectives at various phases of the fermentation process, which open new avenues to better understand fermentation. This study demonstrates that, when coupled with appropriate signal analysis tools and methodologies, the MPS technology offers unparalleled opportunities for real-time monitoring, optimization, and management of industrial-scale fermentation and other food or beverage production processes which are facilitated by microbial activity.

Enhancing Saccharomyces cerevisiae Taxane Biosynthesis and Overcoming Nutritional Stress-Induced Pseudohyphal Growth.

The recent technological advancements in synthetic biology have demonstrated the extensive potential socio-economic benefits at laboratory scale. However, translations of such technologies to industrial scale fermentations remains a major bottleneck. The existence and lack of understanding of the major discrepancies in cultivation conditions between scales often leads to the selection of suboptimal bioprocessing conditions, crippling industrial scale productivity. In this study, strategic design of experiments approaches were coupled with state-of-the-art bioreactor tools to characterize and overcome nutritional stress for the enhanced production of precursors to the blockbuster chemotherapy drug, Taxol, in S. cerevisiae cell factories. The batch-to-batch variation in yeast extract composition was found to trigger nutritional stress at a mini-bioreactor scale, resulting in profound changes in cellular morphology and the inhibition of taxane production. The cells shifted from the typical budding morphology into striking pseudohyphal cells. Doubling initial yeast extract and peptone concentrations (2YP) delayed filamentous growth, and taxane accumulation improved to 108 mg/L. Through coupling a statistical definitive screening design approach with the state-of-the-art high-throughput micro-bioreactors, the total taxane titers were improved a further two-fold, compared to the 2YP culture, to 229 mg/L. Filamentous growth was absent in nutrient-limited microscale cultures, underlining the complex and multifactorial nature of yeast stress responses. Validation of the optimal microscale conditions in 1L bioreactors successfully alleviated nutritional stress and improved the titers to 387 mg/L. Production of the key Taxol precursor, T5αAc, was improved two-fold to 22 mg/L compared to previous maxima. The present study highlights the importance of following an interdisciplinary approach combining synthetic biology and bioprocessing technologies for effective process optimization and scale-up.

Dynamic analysis of flavor properties and microbial communities in Chinese pickled chili pepper (Capsicum frutescens L.): A typical industrial-scale natural fermentation process

Pickled chili pepper (Capsicum frutescens L.), a traditional Chinese fermented vegetable product, is favored by consumers because of its unique flavor. Microbial succession is the main factor affecting the development of the typical flavor of pickled chili pepper. This study investigated the dynamics of flavor changes (mouthfeel sensation, taste properties, and aroma profile) and microbial diversity (bacterial and fungal communities) during the industrial-scale fermentation of pickled chili pepper. Results revealed that during fermentation, the hardness and pungency of pickled chili pepper decreased significantly (P < 0.05), whereas organic acids, free amino acids, and volatiles (especially terpenes, alcohols, and aldehydes) increased significantly (P < 0.05). Lactobacillus and Candida species, specifically, Lactobacillus brevis and Candida sp. F15, dominated in pickled chili pepper. Correlation analysis indicated that the dominant bacterial (Lactobacillus versmoldensis and L. brevis) and fungal (Colletotrichum simmondsii and Cladosporium tenuissimum) species had a high positive correlation with volatile compounds (e.g., aldehydes, terpenes, and esters).

Domain Adaptation and Federated Learning for Ultrasonic Monitoring of Beer Fermentation

Beer fermentation processes are traditionally monitored through sampling and off-line wort density measurements. In-line and on-line sensors would provide real-time data on the fermentation progress whilst minimising human involvement, enabling identification of lagging fermentations or prediction of ethanol production end points. Ultrasonic sensors have previously been used for in-line and on-line fermentation monitoring and are increasingly being combined with machine learning models to interpret the sensor measurements. However, fermentation processes typically last many days and so impose a significant time investment to collect data from a sufficient number of batches for machine learning model training. This expenditure of effort must be multiplied if different fermentation processes must be monitored, such as varying formulations in craft breweries. In this work, three methodologies are evaluated to use previously collected ultrasonic sensor data from laboratory scale fermentations to improve machine learning model accuracy on an industrial scale fermentation process. These methodologies include training models on both domains simultaneously, training models in a federated learning strategy to preserve data privacy, and fine-tuning the best performing models on the industrial scale data. All methodologies provided increased prediction accuracy compared with training based solely on the industrial fermentation data. The federated learning methodology performed best, achieving higher accuracy for 14 out of 16 machine learning tasks compared with the base case model.

Microbial dynamics in industrial-scale wine fermentation employing Hanseniaspora uvarum β-glucosidase-producer strain.

The use of non-Saccharomyces yeast strains in winemaking is becoming a common trend. In fact, consumers are demanding new and healthier styles of wine. On the other hand, these strains are a challenge for the starting process due to winery-resident strains, especially with regard to industrial-scale fermentations. Current assay focuses on the scale-up of the laboratorial inoculum inside the winery environment to ferment 15,000 and 25,000 L of Vitis labrusca Bordô must, using a Hanseniaspora uvarum β-glucosidase-producer strain as starter culture. This scale-up could confirm the viability of using non-Saccharomyces yeast, as it presented promising results on a laboratory scale. The non-Saccharomyces strain was selected in a previous study since it proved to increase resveratrol concentration in lab scale winemaking. The yeast diversity was followed by the plate culturing method. Species identification and strain typing were determined by ITS-RFLP and PCR-fingerprinting, respectively. Physical and chemical analyses and resveratrol quantification were performed in the elaborated wines.

Understanding the differences in 2G ethanol fermentative scales through omics data integration.

In this work, we evaluated the fermentative performance and metabolism modifications of a second generation (2G) industrial yeast by comparing an industrial condition during laboratory and industrial scale fermentations. Fermentations were done using industrial lignocellulosic hydrolysate and a synthetic medium containing inhibitors and analyses were carried out through transcriptomics and proteomics of these experimental conditions. We found that fermentation profiles were very similar, but there was an increase in xylose consumption rate during fermentations using synthetic medium when compared to lignocellulosic hydrolysate, likely due to the presence of unknown growth inhibitors contained in the hydrolysate. We also evaluated the bacterial community composition of the industrial fermentation setting and found that the presence of homofermentative and heterofermentative bacteria did not significantly change the performance of yeast fermentation. In parallel, temporal differentially expressed genes (tDEG) showed differences in gene expression profiles between compared conditions, including heat shocks and the presence of up-regulated genes from the TCA cycle during anaerobic xylose fermentation. Thus, we indicate HMF as a possible electron acceptor in this rapid respiratory process performed by yeast, in addition to demonstrating the importance of culture medium for the performance of yeast within industrial fermentation processes, highlighting the uniquenesses according to scales.

Real-time and on-line monitoring of ethanol fermentation process by viable cell sensor and electronic nose

In this study, introduction of a viable cell sensor and electronic nose into ethanol fermentation was investigated, which could be used in real-time and on-line monitoring of the amount of living cells and product content, respectively. Compared to the conventional off-line biomass determination, the capacitance value exhibited a completely consistent trend with colony forming units, indicating that the capacitance value could reflect the living cells in the fermentation broth. On the other hand, in comparison to the results of off-line determination by high-performance liquid chromatography, the ethanol concentration measured by electronic nose presented an excellent consistency, so as to realize the on-line monitoring during the whole process. On this basis, a dynamic feeding strategy of glucose guided by the changes of living cells and ethanol content was developed. And consequently, the ethanol concentration, productivity and yield were enhanced by 15.4%, 15.9% and 9.0%, respectively. The advanced sensors adopted herein to monitor the key parameters of ethanol fermentation process could be readily extended to an industrial scale and other similar fermentation processes.

CRISPR/Cas-based genome editing in Aspergillus niger

Aspergillus niger is a vital industrial workhouse widely used for the production of organic acids and industrial enzymes. This fungus is a crucial cell factory due to its innate tolerance to a diverse range of abiotic conditions, high production titres, robust growth during industrial scale fermentation, and status as a generally recognized as safe (GRAS) organism. Rapid development of synthetic biology and systems biology not only offer powerful approaches to unveil the molecular mechanisms of A. niger productivity, but also provide more new strategies to construct and optimize the A. niger cell factory. As a new generation of genome editing technology, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) system brings a revolutionary breakthrough in targeted genome modification for A. niger. In this review, we focus on current advances to the CRISPR/Cas genome editing toolbox, its application on gene modification and gene expression regulation in this fungal. Moreover, the future directions of CRISPR/Cas genome editing in A. niger are highlighted.