Microbes in Action: Powering Sustainable Fermentation for Food, Pharma, and Bioeconomy

Abstract

Background and Objective: Fermentation, a microbial-driven metabolic process, has been utilized for millennia to produce, preserve, and enhance food and beverages, evolving from traditional practices such as yogurt and wine to modern applications in biofuels and pharmaceuticals. Fermentation has been found to have its earliest evidence more than 7,000 years ago. The principle of fermentation was established in the 19th century, based on the foundational research of Pasteur, who bridged the connection between microbial activity and chemical change, setting the stage for modern microbiology and biotechnology. This review explores microbial roles in fermentation, bridging traditional and biotechnological advancements by comparing bacterial and fungal processes, analyzing key metabolites, and highlighting genetic innovations. Results and Conclusion: The review comprehensively explores the pivotal role of microbial fermentation, spanning traditional practices to modern innovations, highlighting the extensive diversity of 195 bacterial and 69 fungal species, with lactic acid bacteria (LAB) and Saccharomyces cerevisiae being prominent examples. It compares bacterial and fungal fermentation processes, noting that bacterial fermentation often yields higher protein content in products like fermented soybean meal (FSBM), and discusses key metabolites, including primary (amino acids, organic acids, vitamins) and secondary (antibiotics, antitumor agents) compounds, for their industrial and health applications. The review also examines various fermentation methods and their suitability for different products, emphasizing advancements in genetic engineering for strain optimization, while underscoring the health benefits of probiotics and fermented foods and the potential of emerging technologies to address food security and sustainability. In conclusion, microbial fermentation bridges ancient traditions with cutting-edge science, offering transformative potential across industries, where innovations in genetic engineering and process optimization drive efficiency and sustainability, and the expanding microbial repertoire continues to unlock novel applications, integrating fermentation technology with modern biotechnological tools to address global challenges in nutrition, health, and environmental sustainability. Keywords: Biofuel production, bioeconomy, CRISPR-Cas9, food security, probiotics, solid-state fermentation (SSF), submerged fermentation (SmF), sustainability, synthetic biology. Microorganisms have played a central role in human life, particularly during the food fermentation process. Fermentation has been utilised over the centuries to produce, conserve, and flavour beverages and foods, enhancing their nutritional and sensory qualities. Fermentation is a metabolic process that uses microorganisms to break down carbohydrates into alcohol, organic acids, gases, or other desired by-products [1]. The fermentation processes were induced by naturally occurring populations of bacteria, yeasts, and moulds, whose synergistic activity produced characteristic textures, flavours, and nutritionally beneficial advantages. This has evolved from a mystical art to a science with the discovery of microorganisms by pioneers like Antonie van Leeuwenhoek and Louis Pasteur [2]. Development of microbiology, molecular biology, and bioprocess engineering has made it possible to identify, isolate, and genetically improve microbial strains for large-scale fermentations. Fermentation has numerous applications in the biotechnology industry, including the production of antibiotics, vaccines, enzymes, biofuels, biodegradable plastics, and food products. Considering recent health and sustainability concerns, microbial fermentation offers promising routes for producing plant-based meat alternatives, upcycling agro-industrial waste into high-value products, and generating nutraceuticals and pharmaceuticals. Probiotic fermentation is another contemporary area of research in food fermentation. It refers to the process where live microorganisms, known as probiotics, are introduced to a food or substrate to undergo fermentation, resulting in a variety of beneficial changes [3]. This process enhances the food's nutritional value, digestibility, and flavour, while also introducing beneficial bacteria that can positively impact the consumer's gut health [4]. Probiotic technology leads the functional food sector based on strain specificity, gut microbiota interaction, and individualised nutrition. Contamination control issues, regulatory acceptance of genetically modified strains, consumer acceptability, and scaling up of new technology are being addressed more effectively through interdisciplinary and collaborative partnerships at a global level [5]. The function of microbes in fermentation is a balance between tradition and innovation, promoting an interrelated and symbiotic collaboration between human creativity and microbial activity. Fermentation is a biotechnology that has been utilised for millennia to flavour and preserve food, produce drinks, and create pharmaceuticals. Its roots date back to microbes that transform raw material into staple foods, such as bread, cheese, wine, and beer [2]. Fermentation is coupled with technology to produce a wide variety of products, ranging from traditional fermented foods and specialty beverages on one end and antibiotics, biofuels, and recombinant proteins on the other [6].  The metabolic diversity of microbes has been utilised to produce both submerged and solid-state fermentation systems, each offering distinct advantages for specific applications. The introduction of synthetic biology, genetic engineering, and omics technologies has further transformed microbial fermentation with improved metabolic pathways for precision biomanufacturing. Technologies such as continuous fermentation, immobilised cell systems, and electro-fermentation have enhanced process efficiency and sustainability. Additionally, their applications in cellular agriculture, biodegradable plastics, and waste valorisation reflect the ability of fermentation to address key challenges in food security, healthcare, and environmental sustainability. Fermentation is alternatively referred to as Zymology or Zymurgy and was practised by humans since the Neolithic era (around 10000 BC). Industrial fermentation involves the use of microorganisms, such as bacteria and fungi, to produce goods beneficial to humans and acidic products with applications in the food industry [7]. Fermentation rate depends on the concentration of microorganisms, cell types, cellular constituents, enzymes, temperature, pH, and the aerobic fermentation factor. Inventory of microbial species used in fermentation Over the past two decades, the number of microbial species used in fermentation processes has significantly increased. The 2002 International Dairy Federation Inventory, which catalogued 82 bacterial species and 31 yeast and mould species, has been replaced by the current Inventory of Microbial Food Cultures, which now includes 195 bacterial species and 69 yeast and mould species [8]. This growth is attributed to advanced genomic sequencing techniques, increased demand for a variety of fermented foods, and advancements in microbial biotechnology. The bacterial repository has experienced a meteoric rise in lactic acid bacteria (LAB) genera, with lactobacilli comprising 45 different species used in dairy, meat, vegetable, and cereal fermentations [8]. In 2020, the Lactobacillus genus was significantly reorganized, with many species being reclassified into new genera. This included the creation of 23 new genera. For example, Lactobacillus casei is now Lacticaseibacillus casei, and Lactobacillus plantarum is now Lactiplantibacillus plantarum. Lactobacillus rhamnosus is now Lacticaseibacillus rhamnosus, and Lactobacillus brevis is now Levilactobacillus brevis. Lactobacillus salivarius is now Ligilactobacillus salivarius, and Lactobacillus fermentum is now Limosilactobacillus fermentum [9]. The number of Bifidobacterium species increased from 4 to 10 due to their importance in probiotic applications. The yeast and mould inventory has grown from traditional Saccharomyces cerevisiae and Aspergillus oryzae species to include a broad range of non-traditional species such as Pichia kudriavzevii for cocoa fermentation and Neurospora intermedia for oncom production. The identification and utilisation of extremophiles, such as thermophilic Geobacillus species in hot fermentations and halophilic Archaea in high-salt processes, has triggered diversification of microorganisms. It also includes engineered strains developed through the application of both traditional mutagenesis and novel genetic techniques, such as lactose-positive yeast strains for use in dairy applications and bacteriocin-producing LAB variants for enhanced food safety. Regulatory reforms and enhanced safety assessment procedures have enabled the inclusion of previously underutilised species. Concurrently, market demand for flavor, texture, and health-affecting effects has spurred the utilization of microbial diversity from globally dispersed traditional fermented foods [9]. The diversity of microorganisms involved in fermentation processes spans a wide range of taxonomic groups, including bacteria, yeasts, and filamentous fungi. These microbes play pivotal roles in transforming raw substrates into valuable fermented products. As illustrated in Figure 1, the taxonomic distribution highlights the predominance of LAB and Saccharomyces cerevisiae in traditional and industrial fermentations, alongside other key genera such as Aspergillus sp. and Bifidobacterium sp. This microbial richness underscores the versatility of fermentation technology across food, pharmaceutical, and bioeconomic sectors. The distribution of microbial species used in fermentation was systematically categorised across relevant taxonomic units, as detailed in Table 1 (bacteria) and Table 2 (fungi/yeasts). Table 1 highlights the predominance of lactobacilli (45 species) and Bifidobacterium (10 species) among LAB, alongside Bacillus (over 30 species), which are also utilised for industrial enzymes. Table 2 emphasizes Aspergillus and Saccharomyces as key fungal genera, with Aspergillus niger for organic acids and Saccharomyces cerevisiae for ethanol. Some taxa, such as Acetobacter (vinegar) and Pichia (recombinant proteins), reflect diversification. This structured classification shows the microbial richness harnessed in traditional and modern fermentation biotechnologies. 2.1. Bacteria 2.1.1. Actinobacteriaceae The microbial richness in the manufacture of fermented foods has undergone extensive taxonomic resolution and growth, indicative of advances in genomic research for the microbial ecology within food substrates. The genus Brachybacterium is a component of the surface microbiota of artisanal cheeses, such as Gruyère and Beaufort. It plays a role in proteolysis and flavour enhancement through its enzyme activities. Microbacterium gubbeenense is a significant member of the classical red-smear surface cultures employed in surface-ripened cheeses, playing a role in colour development and aroma profile [29]. The genus Bifidobacterium has undergone taxonomic revisions, with the redescription of Bifidobacterium infantis and the addition of Bifidobacterium thermophilum due to its significant roles in functional food applications for probiotic dairy products. Brevibacterium aurantiacum is utilised for its essential role in the ripening process, contributing to the flavour complexity and the distinctive orange colour of such cheeses. The Propionibacterium genus, including Propionibacterium freudenreichii subsp. globosum and Propionibacterium jensenii (now reclassified as Acidipropionibacterium jensenii) [30]. 2.1.2. Firmicutes The International Molecular Foods inventory has expanded significantly with advances in microbial taxonomy and the growing industrial demand for specialised starter cultures in a diverse range of food matrices. Some notable additions include three species of Carnobacterium, Tetragenococcus, Weissella, Enterococcus faecalis, Lactobacillus, Staphylococcus, and Streptococcus. Carnobacterium is essential for meat fermentations, and Tetragenococcus comprises species required for high-salt fermentations, such as those used in the production of soy sauce and fish products. Weissella splits species previously classified as part of the Leuconostoc mesenteroides complex for their distinctive phylogenetic and metabolic characteristics [31]. Enterococcus faecalis is the starter for dairy, meat, and vegetable fermentations, whereas the lactobacilli comprise 82 species. The Staphylococcus genus contains 13 species, which prove their critical contribution towards meat fermentation. This also allows for the specific selection of strains to achieve goal-directed fermentation results, such as balanced acidification and aroma formation in sausages or maximum gas formation in sourdough. 2.1.3. Proteobacteriaceae The International Molecular Foods stock has been supplemented with specialised microbial species, which cover conventional fermentation practices and new biotechnological developments. Acetic acid bacteria, such as Acetobacter sp. and Gluconacetobacter sp., play a crucial role in the production of vinegar, as well as in the fermentation of cocoa and coffee, and in imparting flavour sensations. Halomonas elongata is a halotolerant organism that has been indicated in meat fermentation systems due to its resistance to high sugar levels and acidic environments [32]. This expanded microbiota enables specificity in the fermentation process, as observed in Acetobacter pasteurianus strains used to produce artisanal balsamic vinegar. Zymomonas mobilis, due to its novel pyruvate decarboxylase and alcohol dehydrogenase II pathway, prefers the irreversible production of ethanol. The industrial application of these microbial resources has demonstrated quantifiable effects, wherein Gluconacetobacter europaeus strains reduced vinegar-making times by 30–40 % and specific Zymomonas mobilis isolates achieved 15–20 % productivity gains in ethanol production, making fermentation more feasible for tequila production.   2.2. Fungi 2.2.1. Yeast Fungal taxonomy employed in food production has been extensively utilised, as numerous species formerly associated with the genus Candida have been reassigned to more phylogenetically accurate genera. This has significant implications for food science and industry, enabling more accurate strain selection, process optimisation during fermentation, product uniformity, and the prevention of spoilage risk [33]. Examples are Dekkera bruxellensis, Debaryomyces hansenii, Hanseniaspora uvarum, Kazachstania turicensis, Metschnikowia pulcherrima, Pichia occidentalis, Rhodosporidium spp., Saccharomyces pastorianus, Saccharomycopsis fibuligera, Saturnisporus saitoi, Sporobolomyces roseus, Torulaspora delbrueckii, Trichosporon cutaneum, Wickerhamomyces anomalus, Yarrowia lipolytica, Zygosaccharomyces bailii, and Zygosaccharomyces rouxii. The persistence of some Candida species with unknown teleomorphs of fungal life cycles, to explain their sexual phases or validate their taxonomic position using genomic information. The functional properties of these microorganisms are being utilised increasingly, such as Metschnikowia pulcherrima's use in biocontrol to inhibit moulds on vineyards and Wickerhamomyces anomalus's delivery of killer toxins that inhibit spoilage yeasts in fermented foods [34]. 2.2.2. Filamentous fungi Fungi are well-established in Asian food fermentation traditions, and have been added to the list of microorganisms used in fermented foods in Europe. There is significant potential in adopting well-established fungal starter cultures from Asian food fermentation traditions and incorporating them into European practices. Key fungi include Aspergillus oryzae, Aspergillus sojae, and Rhizopus oligosporus, which are essential to produce Asian fermented foods and beverages such as miso, soy sauce, sake, awamori liquors, and Puerh tea. Fusarium species, such as Fusarium domesticum and Fusarium solani, are utilised in European food applications, including cheese fermentations, Vacherin cheese production, and the development of meat alternatives. Penicillium species dominate fungal applications in European dairy traditions, with Penicillium camemberti being used in bloomy-rind cheeses such as Camembert and Brie. European charcuterie traditions utilise Penicillium nalgiovense and select strains of Penicillium chrysogenum for mould-fermented sausages, while Lecanicillium lecanii shows promise for cheese ripening applications. Fungal contributions beyond fermentation to include colourant production, with species like Epicoccum nigrum and Penicillium purpurogenum capable of generating natural pigments [35]. This fungal application highlights the rich diversity of fungal species in global food systems and the significant opportunities for cross-cultural transfer of fungal technologies in Asian fermentation fungi to European practices for novel product development. Comparison of bacterial and fungal Fermentation Fermented Soybean Meal (FSBM) is a highly nutritious protein source, thanks to the unique metabolic pathways and enzymatic activities of the microorganisms involved. Both fungal and bacterial fermentation processes effectively reduce antinutritional factors and enhance the nutritional quality of the resulting products. However, there are notable differences in specific components that influence the suitability of FSBM for various dietary and health applications [36]. Both the fungal and bacterial fermentations increased crude protein content through microbial biomass accumulation, with fungal-fermented FSBM showing a 19.4% increase in soluble protein, a crucial indicator of digestibility, and a more substantial 63.11% increase in bacterial-fermented FSBM. These fermentations also reduced the immunoreactivity of soybean meal, which is essential for minimising allergic responses and enhancing food safety. Amino acid profiles show that crucial amino acids generally remain stable during fermentation, but specific changes occur depending on the microorganism used. These findings have practical implications for animal feed and human nutrition, where FSBM is increasingly used as a high-quality protein source. A comparative parameter of bacterial and fungal fermentation processes is shown in Table 3. Metabolites produced by micro-organisms Microorganisms play a crucial role in fermentation processes, generating diverse metabolites that have been utilized for centuries in food preservation and have evolved into high-tech biotechnological applications [46]. Primary metabolites, including amino acids, nucleotides, vitamins, organic acids, and alcohols, are generated during the growing stage of microorganisms and are of industrial importance. Genetically modified strains, such as Corynebacterium glutamicum and Brevibacterium species, maximise the yield of these metabolites. Secondary metabolites, such as antibiotics, antitumor compounds, and immunosuppressants, are produced during the stationary phase of microbial cultures and have had a profound impact on medicine and agriculture [46]. Downstream processing and fermentation technology enable the effective extraction and purification of these metabolites, facilitating their commercial exploitation. The green advantages of microbial fermentation are being leveraged in global initiatives to achieve a circular bioeconomy. Microorganisms play a crucial role in fermentation processes, generating diverse metabolites that have been utilized for centuries in food preservation and have evolved into high-tech biotechnological applications [46]. Primary metabolites, including amino acids, nucleotides, vitamins, organic acids, and alcohols, are generated during the growing stage of microorganisms and are of industrial importance (Figure 2). Genetically modified strains, such as Corynebacterium glutamicum and Brevibacterium species, maximise the yield of these metabolites. Secondary metabolites, such as antibiotics, antitumor compounds, and immunosuppressants, are produced during the stationary phase of microbial cultures and have had a profound impact on medicine and agriculture [46]. 4.1. Primary metabolites Primary metabolites are small, essential molecules found in all living cells that serve as intermediates or end products of metabolic processes. Primary metabolites comprise amino acids, nucleotides, vitamins, solvents, and organic acids, with applications in industry ranging from human and animal nutrition to the production of biofuels and solvents. Innovative applications include the use of monosodium glutamate and nucleotides in food processing, as well as organic acids such as ethylenediaminetetraacetic acid in pharmaceuticals and water treatment. Additionally, bio-based organic acids like succinic acid are utilised in green packaging and textile production [47]. The production of these metabolites at an industrial scale largely depends on microbial fermentation, where microorganisms are genetically and physiologically engineered to overproduce target molecules through sophisticated biotechnological measures. Technological advancements in bioreactor design and downstream processing methods have further enhanced fermentation processes, with the inclusion of systems biology tools providing a systemic view of cellular metabolism and identifying primary genetic targets for manipulation. The transition to a sustainable and circular bioeconomy has raised demand to produce metabolites using low-value, renewa