Togo Yamada, Pamella Apriliana, Prihardi Kahar
et al.
3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a non-proteinogenic aromatic compound that functions as a key biosynthetic precursor for diverse secondary metabolites with pharmaceutical and industrial value. Microbial production of 3,4-AHBA offers a sustainable alternative to petroleum-based chemical synthesis; however, metabolic complexity and trade-offs between growth and product formation constrain rational strain design. Here, genome-scale metabolic (GSM) modeling and flux balance analysis (FBA) were integrated with targeted genetic engineering to elucidate and enhance 3,4-AHBA production in <i>Streptomyces thermoviolaceus</i>. A genome-scale metabolic model was constructed and expanded by incorporating the <i>nspH–nspI</i> gene operon, which encodes the 3,4-AHBA biosynthetic pathway. In silico FBA predicted substantial rewiring of central carbon metabolism, with carbon flux redirected from glycolysis and the tricarboxylic acid cycle toward aspartate-derived intermediates and 3,4-AHBA synthesis, accompanied by reduced biomass-associated flux. Guided by these predictions, an engineered strain (<i>St::NspHI</i>) was developed and experimentally evaluated. Consistent with model predictions, the engineered strain exhibited lower growth rates and glucose uptake than the wild type, reflecting a metabolic burden. Nevertheless, 3,4-AHBA production was achieved exclusively in the engineered strain. Comparison of simulated and experimental fluxes revealed overestimation by FBA, likely due to secondary metabolism and incomplete genome annotation. Overall, GSM-guided design enables optimization of precursor production.
Fermentation is a type of biological process conducted domestically or commercially to preserve foods and beverages, produce alcohol, add nutritional value and improve aroma and flavor. The natural fermentation of flour in water to obtain a leaven for baking, lately scrutinized in the laboratory with the application of metagenomic methods, has been ubiquitous since the dawn of civilization. Commercially, single culture or defined mixtures of microorganisms are used for their predictability, but regularly fed two-domain microorganism cultures are favored in less industrialized and domestic operations. Fungi principally produce the carbon dioxide responsible for leavening. The bacteria produce acid in the bread commonly known as sourdough for its aroma and flavor. A leaven made by fermentation using flour and water can be stored while it is dormant. We studied a mature culture that is fed twenty-fold with water and flour by incubating it for 24 h, sampling it regularly for pH measurements, and plating it. The colonies were suspended for micrography and DNA extraction for PCR and Sanger sequencing. The metagenomic DNAs were analyzed for bacterial and fungal composition. The proportions of the plant and microbial DNA endogenous to the flour decline rapidly, and the predominant bacteria and fungi in mature leaven propagate, without overlap between the respective microbiomes.
This study examines ancient Chinese brewing origins through archaeological evidence and scientific analysis. Two theoretical frameworks exist: alcohol as an agricultural surplus byproduct versus “Feasting Theory,” which suggests brewing demands stimulated agricultural development. Analysis of pottery residues from the Jiahu site in Wuyang, Henan Province, confirmed mixed fermented beverages from rice, honey, and fruits dating to approximately 9,000 years ago. Since gathering and hunting were the primary subsistence strategies, this demonstrates agriculture was not a prerequisite for brewing. Analysis of yeast cells, starch granules, and phytoliths elucidated brewing materials and fermentation agents. Brewing vessels emerged during the Neolithic period, with the industry establishing during Longshan culture. Grain-based brewing became widespread in the Xia-Shang period, evidenced by artificially cultivated yeast at Shang workshops. Brewing technology advanced during Western Zhou and Spring-Autumn-Warring States periods with specialized administrative positions, and distilled spirits production commenced after the Han dynasty. Alcohol served political, religious, and ritual functions. Dawenkou culture drinking vessels suggest specialized brewers, while Xia-Shang bronze wine vessels and oracle bone inscriptions reflect elite consumption. Western Zhou bronze inscriptions document communal drinking practices and strategic vessel deployment. Changes in bronze wine vessel burial assemblages during late Shang through early-middle Western Zhou reflect ritualization, embodying patriarchal clan system social order. Alcohol maintained social cohesion and institutionalized political authority. Prehistoric consumption is interpreted through costly signaling theory as competitive feasting whereby elites displayed authority. This research demonstrates Chinese brewing culture emerged not necessarily from agricultural development but was feasible during gathering economy stages, suggesting brewing demands may have stimulated agricultural development in certain regions. Brewing technology developed progressively from the Neolithic period, becoming a state-level regulatory system after Western Zhou. Chinese brewing culture became a fundamental component of civilization, functioning as both political instrument and cultural symbol
Jorge Luíz Silveira Sonego, Jaqueline Machado de Moraes, Nayana Simon de Vargas
et al.
Fermentation at high temperatures may be a viable alternative for ethanol production, especially in tropical climate regions. This work describes the evaluation of ethanol production through extractive fermentation at high temperatures using thermotolerant <i>Kluyveromyces marxianus</i>. An experimental design was applied to assess the effect of temperature on the ethanol removal process by CO<sub>2</sub> stripping. Subsequently, kinetic modeling of conventional batch ethanol fermentation at high temperatures was performed, and the hybrid Andrews−Levenspiel model was found to be suitable for describing the kinetics of this process. Experiments were conducted to evaluate ethanol production at high temperatures using thermotolerant yeast, specifically evaluating the effects of different specific CO<sub>2</sub> flow rates (ϕ = 1.0, 1.5, and 2.0 vvm) on ethanol stripping. The results indicated that in all the extractive fermentations conducted with <i>K. marxianus</i>, there was faster substrate uptake and earlier substrate exhaustion compared to conventional fermentation. Significant ethanol removal by stripping was achieved using a CO<sub>2</sub> flow rate of 1.0 vvm (EF<sub>HT</sub>1), and complete substrate consumption was observed by the end of 12 h of fermentation. This result highlights the positive effect of temperature on ethanol entrainment. In addition, integrating the CO<sub>2</sub> stripping technique with high-temperature fermentation (T = 40 °C) improves process efficiency with a lower gas flow rate. This is advantageous, especially for industrial-scale applications, as it can reduce equipment costs associated with the CO<sub>2</sub> feed.
Sara Mitri, Nicolas Louka, Tristan Rossignol
et al.
The bioproduction of 2-phenylethanol (2-PE), a high-value aromatic compound widely used in the fragrance, cosmetic, food and beverage, and pharmaceutical industries, through yeast fermentation offers a sustainable alternative to chemical synthesis and rose extraction. This study explores the fermentation of <i>Yarrowia lipolytica</i> strains using mixed agro-industrial by-products as substrates to produce 2-PE via de novo synthesis, without supplementation with the costly precursor L-phenylalanine. <i>Y. lipolytica</i> strains were genetically engineered to enhance flux through the shikimate pathway and enable the hydrolysis of a broader range of substrates. The culture media consisted solely of a mixture of agro-industrial by-products: sugar beet molasses (SBM), brewer’s spent grain (BSG) pressing extract, and chicory root (CR) pressing extract, serving as the primary carbon and nitrogen sources without the addition of nutrients, minerals, synthetic, complex ingredients, or costly additives. The co-culture approach enhanced substrate utilization, leading to an increase in 2-PE titers, reaching approximately 2.5 g/L 2-PE production after 240 h of fermentation. This study demonstrates the feasibility of integrating co-culture fermentation and agro-industrial waste valorization for sustainable 2-PE production, offering a scalable bioprocess for industrial applications.
Marcelo Luis Kuhn Marchioro, Gabrielli Aline Pietro Bom Candeia, Luana Malaquias Bertoleti
et al.
In response to the growing demand for alternative protein sources and functional biomolecules for industrial applications, this study investigated the production of mycoprotein and extracellular (1→6)-β-D-glucan (lasiodiplodan) by the fungus <i>Lasiodiplodia theobromae</i> MMPI, establishing an integrated biotechnological platform. Soybean molasses were evaluated as a low-cost fermentation substrate and compared to a sucrose-based medium. The experimental design and response surface methodology defined conditions that maximized mycelial biomass and lasiodiplodan production. Mycelial biomass from soybean molasses was rich in essential amino acids, lipids, and polyunsaturated fatty acids like gamma-linolenic and alpha-linolenic acids, while sucrose favored higher lasiodiplodan production. Antioxidant compounds like gallic acid and catechin were also found in the biomass, showing potential for scavenging free radicals. Soybean molasses promoted lipid-rich biomass, suggesting <i>L. theobromae</i> MMPI’s potential for biofuel production. This study highlights the fungus’ utility in producing mycoproteins, lipids, and lasiodiplodan for food, animal feed, and industrial uses.
This study explores the potential of enriching beer with protein by incorporating sweet whey, a by-product of the dairy industry, into the brewing process. With Romania’s high beer consumption, this approach aligns with current consumer preferences for functional and nutritionally enhanced beverages. Sweet whey was added to beer wort at 10%, 15%, and 20% concentrations before fermentation. Four samples were analyzed for their physicochemical properties on the day of preparation and after seven days of fermentation at 18 °C. Parameters such as alcohol content, extract, density, Brix, cryoscopy point, and protein content were assessed. Results showed a positive correlation between whey concentration and fizico-chemical analyses. The findings highlight whey’s valorization potential in brewing and its role in advancing sustainable, functional food products.
Federica Barbieri, Chiara Montanari, Chiara Angelucci
et al.
The use of starter cultures in the meat industry is common, even if the number of available commercial cultures is limited, inducing product standardisation and microbial diversity reduction. On the other hand, some artisanal products relying on spontaneous fermentation can represent a source of isolation of new interesting strains. In this work, four LAB strains derived from Mediterranean spontaneously fermented sausages were tested as new starter cultures for the industrial production of fermented sausages, in comparison to a commercial starter culture. The products obtained were analysed for physico-chemical parameters, microbiota, biogenic amines and aroma profile. A consumer test was also performed to evaluate product acceptability. The strains induced different acidification kinetics. LAB counts showed high persistence when <i>Latilactobacillus curvatus</i> HNS55 was used as the starter culture, while the addition of <i>Companilactobacillus alimentarius</i> CB22 resulted in a high concentration of enterococci (6 log CFU/g), 2 log higher than in other samples. Tyramine was detected at concentrations of 150–200 mg/kg, except for in the sample produced with <i>Lactiplantibacillus plantarum</i> BPF2 (60 mg/kg). Differences were observed in the aroma profile, with a high amount of 2-butanone found in the samples obtained with <i>Comp. alimentarius</i> CB22. These latter sausages also showed the lowest score in terms of acceptability. This study allowed us to select new LAB strains for fermented sausage starter cultures, increasing the product diversification.
The reasons why it is practically impossible to maintain optimal conditions for the development of cultural yeast populations under production conditions are briefly substantiated. A simplified classification of yeast activation methods is given: chemical, physical, and combined. In each of the mentioned groups, the varieties of the proposed technological methods and the modes of their implementation are considered. Experimental data obtained in recent years on the influence of the sound in the audible range (20–20,000 Hz) and light in the visible range on the development of Saccharomyces cerevisiae yeast used in brewing are presented. An attempt made to compare the effectiveness of various ways to improve technological indicators: the increase in the total titer of cells, the percentage of nonviable cells, the accumulation of ethyl alcohol.
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid β-peptide (Aβ) and subsequent oxidative inflammatory response, leading to brain damage and memory loss. This study explores the potential of <i>Antrodia cinnamomea</i> (AC), a Taiwan-native fungus known for its anti-inflammatory and antioxidant properties. The metabolites of AC, including dehydroeburicoic acid (DEA), 4-acetylantroquinonol B (4-AAQB), dehydrosulphurenic acid (DSA), and polysaccharides, were of particular interest. In the experiment, deep ocean water (DOW) was used to facilitate the solid-state fermentation of <i>Antrodia cinnamomea</i> NTTU 206 (D-AC), aiming to enhance its functional components. The impact of D-AC on the modulation of AD-related risk factors and the augmentation of cognitive abilities was subsequently evaluated in an AD rat model. This model was established via consecutive infusions of Aβ40 into the brain over a 28-day period. The administration of D-AC resulted in remarkable improvements in the rats’ reference memory, spatial probe test, and working memory. Notably, it restored the hippocampal magnesium levels by upregulating the expression of the magnesium transporter MAGT1. Concurrently, D-AC significantly downregulated the expressions of β-secretase 1 (BACE1) and the phosphorylated tau protein (p-tau), which were both implicated in AD progression. Additionally, it mitigated inflammatory responses, as suggested by the decreased levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in the hippocampus and cerebral cortex. Ultimately, the ability of D-AC to restore the brain magnesium levels, attenuate inflammatory responses, and reduce hippocampal Aβ40 deposition led to significant improvements in the cognitive decline of AD rats. D-AC demonstrated a comparable efficacy with its counterpart, AC fruiting bodies (F-AC group), despite their componential differences. This study underscores the potential of D-AC, enriched through fermentation, as a novel dietary strategy for Alzheimer’s disease prevention.
Low biomass yield and nutrient removal efficiency are problems challenging the employment of microorganisms for wastewater remediation. Starch processing effluent (SPE) was used as a fermentation substrate to co-culture <i>Chlorella vulgaris</i> and <i>Rhodotorula glutinis</i> for biofuel feedstock production. Co-culture options were compared, and the optimal conditions were identified. The result shows that microalgae and yeast should be inoculated simultaneously at the beginning of SPE-based fermentation to achieve high biomass yield and the optimal inoculation ratio, light intensity, and temperature should be 2:1, 150 μmol/m<sup>2</sup>/s, and 25 °C, respectively. Under the optimal conditions, the lipid yield of microorganisms was 1.81 g/L and the carbon–conversion ratio reached 82.53% while lipid yield and the carbon–conversion ratio in a monoculture fell in the range of 0.79–0.81 g/L and 55.93–62.61%, respectively. Therefore, compared to the monoculture model, the co-fermentation of <i>Chlorella vulgaris</i> and <i>Rhodotorula glutinis</i> in starch processing effluent could convert nutrients to single-cell oil in a more efficient way. It should be noted that with the reduced concentration of residual organic carbon in effluent and the increased carbon–conversion ratio, co-fermentation of microalgae and yeast can be regarded as a promising and applicable strategy for starch processing effluent remediation and low-cost biofuel feedstock production.
The growing interest in fermented plant-based milks and the availability of starter bacterial cultures, as well as equipment (devices that maintain the temperature at the appropriate level for fermentation, dedicated to use at home) have a resulted in many consumers not only producing plant-based milks at home, but also fermenting them. Moreover, the importance of homemade plant-based foods and beverages (including fermented products) significantly increased in recent years. The fermentation of commercial and homemade almond, oat and soy milks to obtain plant-based yogurt alternatives for fresh consumption was compared regarding microorganisms viability, physicochemical characteristics (pH, viscosity, stability), bioactive compounds changes (proteins, free amino acids, total carbohydrates, reducing sugars, polyphenolics and flavonoids) as well as antioxidant activity (DPPH, ABTS, O<sub>2</sub><sup>−</sup> radicals scavenging activities and reducing power). The results showed that both commercial and homemade plant-based milks can be successfully fermented, ensuring high number of Lactic Acid Bacteria, high content of bioactive ingredients and antioxidant activity. Homemade plant-based milks are an attractive alternative to their commercial counterparts, moreover, considering the content of bioactive ingredients (such as polyphenols and flavonoids) and antioxidant activity, homemade almond, oat and soy milks showed a more favorable profile.
The course of weather conditions during the growing season, but also beyond it, decides the economics of hop cultivation every year. Ongoing climate changes manifested by more frequent occurrence of compound hot and dry waves affect hop cultivation not only in the Czech Republic, but in the entire region of Central Europe. The paper evaluates the harvest results (yields, alpha acid content) of four Czech hop varieties in the period 2019–2022 with a focus on the weather diametrically opposed vintages 2021 and 2022 on a specific agricultural farm in the Žatec (Saaz) hop-growing region. While the 2021 vintage was rich in precipitation in the decisive vegetation phase, the 2022 vintage was very dry and hot. The age of the plants significantly influenced the alpha acid content of the varieties Saaz, Sládek and Premiant independently of the vintage. Young growths of the Saaz variety contained at least 60% rel. more alpha acids than older growths (more than 5 years old), while the increase in the Sládek variety was 30–40% rel. and in the Premiant variety 20–25% rel. In the case of the variety Agnus, the age of the plants did not have a demonstrable effect on the content of alpha acids. In 2021 the yields of all varieties were negatively affected by the calamitous occurrence of downy mildew. In 2022, the rainfall deficit was so large that it could not be fully compensated even by the maximum use of drip irrigation. Of the evaluated varieties, the Agnus variety coped best with the different weather conditions of the assessed vintages. This shows that the cultivation of climate resistant varieties is one of the promising but time-consuming ways to face the expected climate changes. Other options include regular renewal of hop growths, consistent regionalization, i.e. planting hop in locations that are suitable for the given variety and efficient use of irrigation systems.
Nurul Syahirah Mat Aron, Kit Wayne Chew, Zengling Ma
et al.
The increase in global temperature calls for ambitious action to reduce the release of greenhouse gases into the atmosphere. The transportation sector contributes up to 25% of the total emissions released, mainly from the burning of vehicle fuel. Therefore, scientists from all around the world are focusing on finding a sustainable alternative to conventional vehicle fuel. Biofuel has attracted much attention, as it shows great potential for the replacement of traditional fossil fuels. However, the main bottlenecks of biofuel are the ongoing controversial conflict between food security with biofuel production. Therefore, this study focuses on a sustainable extraction of lipids from microalgae for the production of biofuel using a liquid biphasic flotation system coupled with sugaring-out method. This is the first study to combine the methods of liquid biphasic flotation system with the sugaring-out technique. It represents a holistic study of optimum and effective conditions needed to extract lipids from the system and to understand the reliability of sugar solution as the agent of cell disruption. At the 15-min flotation time, 150 g/L of fructose solution with a 1:2 mass separating agent-acetonitrile ratio successfully extracted up to 74% of lipid from <i>Chlorella sorokiniana</i> CY-1. Two types of fatty acid methyl esters were recovered from the study, with C5:0 being the main component extracted.
Indrajeet Yadav, Akhil Rautela, Agendra Gangwar
et al.
A hemiterpene, isoprene, is commercially produced from crude oil refining processes. As a result of fossil fuel depletion, isoprene production process development is gaining attention from recombinant cyanobacteria and other microbial systems for its industrial and biofuel applications. In the present study, a fast-growing and CO<sub>2</sub>-tolerant cyanobacteria, <i>Synechococcus elongatus</i> UTEX 2973, is engineered with <i>Pueraria montana</i> isoprene synthase (<i>IspS</i>) at neutral site I (NSI) in the genome of <i>S. elongatus</i> UTEX 2973. Furthermore, to enhance isoprene production a key enzyme (isopentenyl diphosphate isomerase, <i>IDI</i>) of the methyl-D-erythritol 4-phosphate (MEP) pathway is also overexpressed at neutral site III (NSIII). Wild-type and recombinant strains of <i>S. elongatus</i> UTEX 2973 (UTEX <i>IspS</i> and UTEX <i>IspS.IDI</i>) are studied for growth and isoprene production in the presence of an inducer (IPTG) and/or inhibitor (alendronate). Alendronate is used for the inhibition of geranyl diphosphate synthase (CrtE), downstream of the MEP pathway that catalyzes dimethylallyl diphosphate/isopentenyl pyrophosphate (DMAPP/IPP) condensation in the recombinant UTEX 2973 strains. The docking studies on SeCrtE (CrtE of <i>Synechcoccus elongatus</i> PCC 7942) and alendronate as an inhibitor have revealed that alendronate binds more tightly than IPP in the cavity of SeCrtE, with a higher number of intermolecular interactions and energy. The UTEX <i>IspS</i> strain has shown isoprene production below the limit of detection in the presence of an inducer and/or inhibitor; however, production studies using UTEX <i>IspS</i>.<i>IDI</i> showed a maximum production of 79.97 and 411.51 µg/g dry cell weight (DCW) in a single day in the presence of an inducer only and an inducer along with an inhibitor, respectively. The UTEX <i>IspS</i>.<i>IDI</i> strain produced 0.41 mg/g DCW of cumulative isoprene in the presence of an inducer and 1.92 mg/g DCW in the presence of an inducer as well as an inhibitor during six days of production. The yield improvement of isoprene is observed as being 4.7-fold by using the inhibition strategy, which is used for the first time in the recombinant cyanobacterial system. The average productivities of isoprene obtained from UTEX <i>IspS</i>.<i>IDI</i> are observed to be 2.8 μg/g DCW/h in the presence of an inducer and 13.35 μg/g DCW/h in the presence of an inducer as well as an inhibitor. This study provides a basis for the process development and yield improvement in isoprene production using a novel inhibition strategy in fast-growing recombinant cyanobacteria. Recombinant strains and metabolic pathway inhibition studies can be used in future attempts to photosynthetically produce hemiterpenes.
Marcia Franco, Ilma Tapio, Juho Pirttiniemi
et al.
New technologies related to the identification of bacterial communities in fresh forage and silage may give valuable detailed information on the best practices to produce animal feeds. The objective was to evaluate how management conditions during silage making manipulate the profile of bacterial communities and fermentation quality of grass silages. Silages were prepared from mixed timothy and meadow fescue grass using two compaction levels. As an additional treatment the grass was contaminated with soil and feces prior to tight compaction. Four additive treatments with different modes of action were applied: control without additive, formic acid-based additive, homofermentative lactic acid bacteria and salt-based additive. After 93 days the silos were opened, samples were taken and routinely analyzed. DNA extraction was carried out and PCR amplification of the bacterial 16S rRNA gene V4 region was performed using universal primers. The silage pH was higher for loose than tight compaction and higher for non-contaminated than for contaminated silages. Great shift was observed in bacterial profiles from fresh material towards silage. <i>Lactobacillus</i> genus was barely found on the relative abundance of fresh grass but became predominant in the final silage along with <i>Sphingomonas</i> genus. Use of additives improved fermentation quality and modified the bacterial profiles of grass ensiled under different management conditions.
Tatiana Felix Ferreira, Fernanda Faria Martins, Caroline Alves Cayres
et al.
<i>Yarrowia lipolytica</i> is a unique, strictly aerobic yeast with the ability to degrade efficiently hydrophobic substrates. In the present work, we evaluated the degrading potential of <i>Yarrowia lipolytica</i> IMUFRJ 50682, isolated from tropical estuarine water in Rio de Janeiro (Brazil), and the possible biomolecules produced during this process. To investigate which crude oil compounds are degraded by <i>Y. lipolytica</i> IMUFRJ 50682, this microorganism was grown in a medium containing Marlim petroleum (19 °API, American Petroleum Institute gravity) at 28 °C and 160 rpm for 5 days. The residual petroleum was submitted to gas chromatograph-mass spectrometric analysis (GC-MS). The chromatographic fingerprints of the residual petroleum were compared with the abiotic control test incubated in the same conditions. <i>Y. lipolytica</i> assimilates high molecular weight hydrocarbons, such as n-alkanes (C11-C19), isoprenoids (pristane and phytane), aromatics with two or three aromatics rings (naphthalene, methylnaphthalenes, dimethylnaphthalenes, trimethylnaphthalenes, phenanthrene, methylphenanthrenes, dimethylphenanthrenes, anthracene). This strain was also capable of consuming more complex hydrocarbons, such as tricyclic terpanes. During this biodegradation, the emulsification index of the culture medium increased significantly, showing that biosurfactant molecules can be produced from this process. Therefore, <i>Y. lipolytica</i> IMUFRJ 50682 showed to be a potential crude oil degrading yeast, which can be used for bioremediation processes and simultaneously produce bioproducts of commercial interest.