Microbial Conversion of Inulin to Valuable Products: The Biorefinery Concept

Abstract

The global transition to a sustainable bioeconomy requires the engagement of renewable and cost-effective substrates to obtain valuable bio-based products. Inulin-rich plant materials have promising applications in white biotechnology. This review evaluates the potential of converting inulin through an integrated biorefinery into high-value products by microbial fermentation. It describes the methods for raw biomass and inulin pretreatment, the possibilities of simultaneous saccharification and fermentation (SSF), and the use of wild-type and genetically modified microbial strains. The bioconversion of inulin enables the efficient synthesis of biofuels such as ethanol, butanol, and 2,3-butanediol and biochemicals such as lactic, citric, and poly-γ-glutamic acid. By analyzing the advances in inulin hydrolysis methods, microbial engineering, and bioprocess optimization approaches, this review highlights the broad applicability of inulin in the biorefinery context as a multifunctional, sustainable substrate, which contributes to the development of the circular economy.

1. Introduction

The global economy currently needs a circular and sustainable transition focused on bioenergy. Biofuels and bioproducts are required for energy needs, reducing greenhouse gas emissions, and tackling global warming as a part of the Green Deal to make Europe the first climate-neutral continent [1,2]. Biomass is a renewable source of energy that offers the possibility of turning waste into valuable resources and reducing the environmental impact. A biorefinery unifies the facilities and the relevant processes dedicated to biomass conversion into bioproducts and bioenergy. It uses renewable resources sustainably and efficiently, in a similar way to oil refineries processing crude oil into multiple products. The development of the biorefinery concept aims to replace fossil fuels with alternative sources, reducing greenhouse gas emissions and contributing to the circular economy by transforming biomass into a wide range of valuable products [2,3].

Biorefineries can be classified based on the type of feedstock used. First-generation biorefineries converted edible crops (starch and sucrose-containing), oil seeds, and animal fats; the second lignocellulosic biomass; and the third focuses on algae-based biogas and bioethanol. The fourth generation of biorefinery relies on engineered microorganisms, bacteria, yeast, fungi, microalgae, and cyanobacteria (to photosynthesize and acquire CO₂ into fuel) [4]. The raw materials used define the biorefineries as wood, corn, palm oil, or algae-based. These definitions determine two main types according to the critical products obtained: energy-driven and product-driven. Energy-driven biorefineries focus primarily on producing biofuel and bioenergy, whereas product-driven biorefineries emphasize bioproduct synthesis, such as food, chemicals, and other materials [5,6].

Inulin as a feedstock is attractive for both energy- and product-driven biorefinery systems (Figure 1). It serves as a feedstock for biofuel (ethanol, butanol) and biochemical (organic acids, poly-γ-glutamate) production.

Inulin is a polysaccharide many plants produce to store energy, accumulating mainly in the underground roots and tubers. Inulin is part of a larger group of water-soluble carbohydrate fructans linear glucosyl-α(1→2)-(fructosyl)n-β(2→1) polymers with a degree of polymerization (DP) of 3–60 [3,5,6]. Inulin is widespread and found in over 36,000 plants, thus confirming the versatility and abundance of inulin sources [7]. Tubers of plants of the Asterales order, such as Jerusalem artichoke (Helianthus tuberosus) and chicory (Cichorium intibus), have high inulin content [3]. Other plants that are rich in inulin include garlic [7], asparagus [8], dandelion [9], globe artichoke [10], dahlia [11], and agave [12].

Table 1. Inulin content in different plants (adapted from Singh et al. [11]).

Inulin-Containing Plant	Latin Name	Inulin-Containing Plant Part	% Inulin 1	Reference
Agave	Agave americana, Agave tequilana	Lobes	7.0–12.0	[12]
Asparagus	Asparagus officinalis	Stems	2.0–3.0	[8,13]
Barley	Hordeum vulgare	Seed	0.5–1.5	[14]
Burdock	Arctium sp.	Roots	3.5–4.0	[14]
Chicory	Cichorium intybus L.	Root	14.9–68.0	[15,16]
Dahlia	Dahlia pinnata	Tuber	10–20.0	[13]
Dandelion	Taraxacum officinale L.	Root	12.0–15.0	[11,14]
Garlic	Allium sativum	Cloves	16.6–24.9	[7,14]
Jerusalem artichoke	Helianthus tuberosus L.	Tuber	14.0–23.0	[16]
Leek	Allium ampeloprasum	Bulb	3.0–10.0	[14]
Onion	Allium cepa L.	Bulb	3.1–6.0	[14]
Sweet leaf	Stevia rebaudiana	Root	18.0–23.0 2	[17]
Salsify	Tragopogon porrifolius L.	Root	4.0–11.0	[13]
Yacón	Polymnia sonchifolia	Root	3.1–19.0	[14]

¹ Percentage in dry biomass; ² Based on the inulin content as % of the fresh weight.

shows the inulin content of different plants and their parts.

The perennial herb Yacón (Smallanthus sonchifolius), a plant from the Asteraceae family, stores 3–10% inulin in its tubers. From the Liliaceae family, garlic (Allium sativum) stores carbohydrates mainly in its cloves, comprising about 75% of its dry matter [7]. Asparagus (Asparagus racemosus) contains up to 15% inulin in its roots [8]. Dandelion (Taraxacum officinale), another member of the Asteraceae family, contains 12–15% inulin and oligofructose in its taproots [9]. Artichoke (Cynara scolymus) accumulates about 50–70 g of inulin per kilogram of fresh weight.

The degree of polymerization (DP), which refers to the number of fructose units in the polysaccharide chain, can vary and is influenced by many factors, such as the plant species, its developmental stage, environmental conditions, and storage time after harvest. Dahlia and Cynara scolymus inulin have a higher degree of polymerization, similar to globe thistle (Echinops ritro) and Viguiera discolor. The solubility of inulin-containing flours is dependent on fructose chain length. For example, 100 g/L soluble chicory flour Frutafit^® CLR contains approximately 19.4 g/L of sugars (7.9 g/L of fructose, 1.5 g/L of glucose, and 10 g/L sucrose), while insoluble Frutafit^® HD contains less than 10 g/L of mono- and disaccharides in total [3]. Furthermore, the soluble Frutafit^® CLR contains oligosaccharides with a lower degree of polymerization, mainly DP3, DP4, and DP5 [3].

Jerusalem artichoke has several desirable traits as a crop: tolerance to cold, drought, and saline, and high resistance to wind, sand, pests, and diseases. It can grow in marginal lands like sandy or tideland areas and accumulate 50–70 g/kg of fresh weight, with a crop yield estimated at 5.4 tons/ha. Chicory also inhabits arid regions that are not appropriate for growing food crops. In addition, it has another advantage of being harvested thrice yearly and contains a large amount of inulin—68% in fresh chicory roots, compared to 7–30% in Jerusalem artichoke tubers. Moreover, dried chicory root extract is 98% inulin and has shorter chains than Jerusalem artichoke, with an average DP of around 10–12. This is why chicory inulin is usually more soluble and easily digestible [6].

Regardless of their characteristics, both crops are industrially important, with significant worldwide harvests. According to United Nations Food and Agriculture Organization (FAO) statistics, the area used for artichoke harvesting significantly exceeds that used for chicory (Figure 2a), and the annual world production of artichoke in tons is almost fifty times that of chicory (Figure 2b).

According to a report by Polaris Market Research, the inulin market was valued at USD 1.64 billion in 2023 and will probably rise to USD 2.98 billion by 2032. The annual growth rate (CAGR) will grow by 6.9%. The European region is the largest inulin market, but Asia Pacific will have the highest growth in the coming years.

2. Pretreatment of Inulin-Containing Biomass

2.1. Extraction

Inulin is obtained from natural plant sources using hot water as an extracting solvent [19]. Pretreatment, extraction, and purification are the three main steps in a typical inulin production process [20]. The highest recovery of inulin from the raw material uses water at temperatures ranging from 70 °C to boiling [21,22,23,24]. The pretreatment process of inulin-rich raw materials consists of slicing and grinding them into tiny particles to enhance mass transfer. Aqueous extraction usually follows, using hot water, separating the enriched solvent from the solid residues, and then purification by bleaching, activated carbon adsorption, or ion-exchange resin treatment [19]. Finally, the inulin extract is dried to obtain pure inulin powder. Hot water extraction and moist steam pressure employ a large amount of water and have high energy consumption; therefore, other methods would alleviate this energy and cost-intensive step [25].

2.2. Enzymatic Hydrolysis

Inulin hydrolysis to fermentable sugars occurs through hydrolytic enzymes. Inulinases belong to the glycoside hydrolase 32 (GH32) family and are classified into two main types. Exoinulinases (β-D-fructan fructohydrolase, EC 3.2.1.80) act by sequentially removing terminal fructose units from the non-reducing ends of the inulin chain with fructose as the main product. Endoinulinases (2,1-β-D-fructan fructanohydrolase EC 3.2.1.7) randomly cleave the internal β-2,1-glycosidic bonds of inulin, leading to the production of inulooligosaccharides, which are shorter chains of fructose units. Other enzymes of the same family are fructan beta-(2,1)-fructosidase (EC 3.2.1.153), 1,2-β-fructan 1^F-fructosyltransferase (EC 2.4.1.100), and sucrose 1^F-fructosyltransferase (EC 2.4.1.99), the last two yielding short-chain fructooligosaccharides (FOS) from sucrose by transferring fructose residues onto a sucrose molecule. Exoinulinases, compared to endoinulinases, hydrolyze inulin more thoroughly, producing fructose and glucose, which can then be used in various fermentation processes [26].

Microorganisms secrete extracellular inulinase to utilize inulin as a carbon and energy source. Fungi (Aspergillus, Penicillium), bacteria (Pseudomonas, Bacillus spp.), and yeast such as Kluyveromyces all synthesize extracellular inulinases. These microorganisms are commonly found in natural environments and play an essential role in the bioconversion of inulin into simpler sugars that can be metabolized.

Fungal and yeast strains typically produce higher levels of extracellular inulinase and are suitable for commercial and industrial applications where efficiency and yield are key factors. Two species of Kluyveromyces (K. fragilis and K. marxianus) are known for their high potential to produce commercially viable yields of inulinase. These yeast species are particularly valued in industrial applications [27]. Aspergillus spp., Penicillium spp., Rhizopus spp., Bacillus spp., Clostridium spp., Streptomyces spp., and yeasts of the Saccharomyces and Pichia genera are among the best inulin-hydrolyzing microorganisms. They are easy to culture, have high enzyme yields, and do not produce toxic byproducts [28]. Inulinases have a molecular weight of over 50.0 kDa, an optimal pH range between 4.5–7.0, and a temperature optimum between 30 and 60 °C [29,30].

Microbial inulinase is perfect for saccharifying inulin-containing crops or agricultural waste [31]. Plants like dandelion, chicory, and Jerusalem artichoke also contain inulinase, but its production cost is high due to the difficulty of obtaining the enzyme in large quantities [32]. One key biotechnological aspect of inulin utilization is the development of microbial strains with an enhanced ability to produce inulinase. Engineered microorganisms can produce higher levels of inulinase and reduce the cost of inulin hydrolysis, making the process more economically viable for industrial applications. By optimizing strains for higher inulinase production, biotechnological processes involving inulin can become more productive, efficient, and cost-effective, contributing to the development of sustainable bioprocesses [1,2,3,4].

2.3. Acidic Hydrolysis

Acid hydrolysis is a standard method to achieve fast and cheap inulin conversion into fermentable sugars by breaking down polysaccharides into oligosaccharides and monosaccharides in the presence of dilute or concentrated acids like sulfuric, hydrochloric, phosphoric, etc. To ensure the optimal recovery and decomposition of the polymer into its component monosaccharide units, technological parameters like temperature, acid concentration, and duration of acid hydrolysis are significant and extensively optimized [33]. Concentrated acid hydrolysis occurs at a lower temperature, but the large amount of acid used causes corrosion and environmental hazards. Dilute hydrolysis is preferred over the concentrated acid process due to the equipment’s lower cost and reduced stress (hence lower maintenance cost). The process runs at elevated pressures and temperatures. However, this facilitates the formation of colored byproducts and the production of toxic compounds (such as furfural), inhibiting microbial growth [34].

Zong et al. investigated acid hydrolysis conducted under varied conditions (temperature, acid concentrations, and time) on fructans (Fru) and inulin-type fructans (ITF). With the intensification of hydrolysis conditions, the generation of byproducts (5-hydroxymethyl-2-furaldehyde, 5-methyl-2-furaldehyde, and furfural), closely associated with fructose degradation, increased. Furthermore, this treatment requires an additional detoxification process, which is complex, costly, time-consuming, and environmentally harmful. Enzymatic hydrolysis of inulin is therefore preferred over acidic hydrolysis [35].

2.4. Simultaneous Saccharification and Fermentation (SSF)

Simultaneous enzymatic hydrolysis and fermentation (or Simultaneous Saccharification and Fermentation, SSF) was first proposed by Gauss et al. in a patent from 1976 [36]. SSF is a combination method in which enzymatic hydrolysis and fermentation of saccharified sugars are conducted simultaneously in the same vessels or reactor. As a single-step process for enzymatic hydrolysis and fermentation, it has many advantages, such as reduction of the initial costs, the inhibitory compounds, and the fermentation time [37]. It is essential to find the optimal conditions for the processes to work properly (optimal temperature and pH), as there are different process parameters for the hydrolyzing enzymes and the bioconversion steps [38].

2.5. Consolidated Bioprocessing (CBP)

Consolidated bioprocessing (CBP) is a conversion method that combines pretreatment, saccharification, and fermentation in a single vessel or reactor. Single microorganisms or consortiums perform CBP by producing enzymes hydrolyzing the carbohydrates. Then, the feedstock of fermentable sugars is converted into desired end products (biofuels or other value-added products) [4]. Selecting natural or recombinant microbial strains is critical in designing CBP to produce advanced chemicals and fuels. The primary issue with native microorganisms is their low yield, productivity, and prolonged fermentation time.

Meanwhile, the main challenge in engineered industrial microorganisms intended for CBP lies in reaching a high biocatalyst production rate without affecting productivity [11]. This approach allows the reduction/elimination of raw materials’ pretreatment steps (and associated costs), making the process more industrially viable and environmentally friendly [39]. Optimal substrate utilization and product-forming properties (increased hydrolysis rate and enhanced productivity) are preferred [11].

3. Biofuel Production from Inulin

The demand for fossil fuels is constantly increasing, and the rapid depletion and negative impact of their use on the climate makes renewable energy sources an attractive and irreplaceable alternative. Biofuels are at the forefront of renewables, especially in the transport industry, where governments have regulated biofuels in fuel blends. This, in turn, is increasing focus on improving biofuel production technology, from developing more efficient catalysts to optimizing process parameters to utilizing previously underused feedstocks.

Similarly to biorefineries, biofuels fall into four categories (with a fifth proposed recently), termed “generations” [40].

First-generation biofuels are derived from crops suitable for human consumption, with corn, sugarcane, and soybeans being the most widely used. They have high yields and are easy to process—fermenting carbohydrates (sugar or starch) with minimal pretreatment. Nevertheless, first-generation biofuels pose a serious social problem [41,42].

Second-generation biofuels are also called “advanced” biofuel. These were produced using non-edible biomass (lignocellulosic (wood, roots, crop residues, dedicated biofuel grasses) and animal wastes). They require a pretreatment step (chemical, physical, physicochemical, biological, or a mix) to counter the structural rigidity and complexity of the biomass, as well as to improve conversion efficiency, followed by a hydrolysis step, before being subjected to fermentation to produce bioethanol [41,43,44].

Third-generation biofuels are comparable to second-generation but use algae as feedstock. For example, photothermal catalysis using a biomass-based graphene-like catalyst at room temperature transesterified microalgae lipids to produce biodiesel with a 96.8% yield [45]. When potassium-containing improved biomaterial was included, the biodiesel yield increased to 99.6% [46].

Fourth-generation biofuels employ genetically modified microorganisms to convert CO₂ and sunlight directly into biofuels. The fifth generation of biofuels will rely on the complex genetic modifications of higher autotrophic eukaryotes, such as algae and plants, for improved carbon fixation [40,41,47,48,49].

Inulin-rich raw materials are an effective substrate for producing various biofuels and fuel additives, the most promising of which are the alcohols—ethanol, butanol (n-butanol and isobutanol), and 2,3-butanediol. Inulin saccharification is more accessible and economically viable than starch or lignocellulosic materials (corn stover, bagasse, sawmill discards) [11]. For entire plant biomass utilization processes, including the lignocellulosic fraction, a preliminary delignification is applied through increased lignin solubility. Recent approaches include bio-based eutectic solvents, with delignification rates of up to 92.2% and 88.4% cellulose recovery achieved [50,51]. New supramolecular deep eutectic solvent (SUPRADES) pretreatment contributed to the valorization of all plant biomass components [52]. After lignin removal, the biomass is usually treated with enzyme cocktails containing cellulases, xylanases, and pectinases to obtain fermentable sugars [53].

3.1. Ethanol

Ethanol is one of the most widely used alcohols in industry (as a solvent) and in medicine (as an anesthetic, antiseptic, and antidote for methanol and ethylene glycol poisoning), but its primary use is as fuel (in stoves, lamps, or combustion engines) [54]. It can be obtained by ethylene hydration from petrol, but its production by fermentation by microorganisms has been established for centuries. Sustainable ethanol production from complex carbohydrate feedstock focuses on further development and optimization of hydrolysis, fermentation, and modifying organisms capable of performing both simultaneously. The global production of bioethanol for 2023 has been estimated to be 112 gigalitres (112 × 10⁹ L), with the top three producers being the USA with 53%, Brazil with 28%, and the European Union with 5% [55]. The price of the raw biomass varies greatly depending on the country in which it is grown (for example, growing and harvesting Jerusalem artichoke costs USD 5300/ha in the USA, USD 2900/ha in Canada, and USD 1080/ha in Australia).

The study by Bedzo et al. [56] evaluated different scenarios of a biorefinery based on JAT producing inulooligosaccharides as the main product and biofuels as side-products and concluded that the raw biomass price accounted for only 10% of the cost of the final products. In line with the sustainable biorefinery concept, Song et al. [57] examined the possibility of producing different products simultaneously using a single feedstock JAT. Zhang et al. utilized JAT as a substrate for bioethanol production as they engineered Pichia pastoris X-33/pPICZaA-INU1 to hydrolyze the inulin into fermentable sugars and Saccharomyces sp. W0 to transform them into ethanol. The authors achieved productivity of 0.384 g/g (98.8% utilization of available sugars) and produced 14.6 g EtOH per 100 mL medium. Incorporating the gene responsible for inulinase in Saccharomyces sp. W0 itself allows the yeast to be used for SSF, and by employing a fermentation medium with 50% (w/v) tuber meal, 12.1% of ethanol was obtained with ethanol productivity of 0.319 g/g, converting over 96.3% (w/v) of the sugars after 144 h fermentation [58] (

Table 2. Production of ethanol from inulin-containing feedstocks.

Inulin Source	Pretreatment	Fermentation Mode	Species/Strain	Ethanol (g/L)	Yield (g/g)	Conversion (%)	Productivity (g/L/h)	Reference
JAT	Enzymatic	Batch	S. cerevisiae	18.0	ND 1	71.8	ND	[57]
JAT	Enzymatic	Batch	Saccharomyces sp. W0	14.6	0.384	98.8	ND	[58]
JAT extract (50%)	Enzymatic	SSF	Saccharomyces sp. W0	12.1	0.319	96.3	ND	[58]
JAT powder	No	SSF, FB 3	S. cerevisiae 6525	84.3	0.453	88.6	ND	[59]
Chicory 2	No	CBP 4	S. cerevisiae JZD-InuMKCP	95.19	0.486	95.0	3.20	[60]
JAT	No	CBP 4	S. cerevisiae JZD-InuMKCP	81.76	0.469	91.7	3.13	[60]
JAT	Acidic/Imidazole	Batch	S. cerevisiae	137.4	0.421	92.47	2.38	[61]
D. sansibarensis	Acidic/0.2 N H2SO4	Batch	I. terricola	56.0	0.49	95.0	1.3	[62]
P. kaurabassana	Acidic/0.2 N H2SO4	Batch	I. terricola	35.0	0.49	96.0	0.8	[62]
JAT stalks	Acidic/0.05 N HCl	Batch	S. cerevisiae	32.5	0.49	88–92	ND	[63]
JAT stalks	Acidic/0.05 N HCl	Batch	K. marxianus	32.0	0.49	96–100	ND	[64]

¹ ND, not determined; ² In medium with yeast extract and peptone added; ³ FB, fed-batch, ⁴ CBP, Consolidated bioprocessing.

).

Wang et al. [59] performed a detailed investigation focused on optimizing the process parameters and scalability (from 250 mL flasks up to 5 L agitating fermenter) of bioethanol production using dry powder of JAT as feedstock. The recombinant exo-inulinase-producing S. cerevisiae 6525 gained 84.3 g/L ethanol after 72 h fermentation in a 5 L fermenter with an efficiency of 0.453 g/g (88.6% of the theoretical maximum). Wang et al. [60] modified the wild S. cerevisiae strain to produce endo- and exo-inulinases, thus being able to ferment inulin to ethanol by CBP. Several modified S. cerevisiae strains, capable of utilizing inulin from chicory or JAT powder, were obtained using various bioengineering techniques. Following additional optimization, the most promising S. cerevisiae JZD-InuMKCP produced 95.19 g/L ethanol (95% of the theoretical maximum) with a yield of 0.486 g/g and ethanol productivity of 3.20 g/L/h for 24 h using 200 g/L chicory inulin. The same strain fermented 250 g/L JAT (without any additional nutrients), producing 81.76 g/L ethanol (91.7% of the theoretical maximum) with a yield of 0.469 g/g and productivity of 3.13 g/L/h for 24 h. The designed strain was appropriate for industrial ethanol production from underused biomass and eliminated the need for expensive additional components to the fermentation medium [60].

The study by Zhao et al. [61] proposed an alternative hydrolysis method to the more widely applied acid and enzyme ones—imidazole-based acidic ionic liquid. Following the optimization procedure, the team determined the best conditions for inulin hydrolysis with the ionic liquid (IL) examined—1.0–2.0% IL concentration, 30% inulin concentration, 65–75 °C, and 50 min reaction time. Comparing their results to acid hydrolysis (diluted H₂SO₄) at the same catalytic load, the authors highlighted the reduced reaction time needed for the ILs. They attributed this enhanced activity to the synergistic effect between the imidazole groups and the hydrogen atoms. Furthermore, they developed a kinetic model that predicts the hydrolysis process at various conditions. Finally, following fermentation with S. cerevisiae, an ethanol concentration of 137.4 mL/L was achieved, with a yield of 0.421 g/g, with 92.47% of the sugars being converted to bioethanol at 2.38 g/L/h productivity without any detrimental effect brought by the use of ionic liquid, highlighting the viability of the approach for further study and possible industrial applications [61].

Moshi et al. [62] investigated inulin-rich tubers from Dioscorea sansibarensis and Pyrenacantha kaurabassana as substrates for bioethanol production by Issatchenkia terricola. The analysis showed that 68% of the dry weight comprises carbohydrates (90% inulin) for D. sansibarensis, and 47% (70% inulin) for P. kaurabassana. After performing acid hydrolysis and multi-parameter optimization, the tubers were subjected to H₂SO₄ acid hydrolysis (1% v/w) at 100 °C, 1.5 bars for 1 h, extracting and hydrolyzing 98.6% of the total carbohydrates of D. sansibarensis and 97.5% of P. kaurabassana. After pH correction to pH 6.0, the strain converted the hydrolysates to bioethanol via batch fermentation. D. sansibarensis produced 56 g/L bioethanol with 0.49 g/g yield (95% fermentation efficiency) and 1.3 g/h/L productivity. P. kaurabassana gained 35 g/L bioethanol with 0.49 g/g yield and fermentation efficiency and productivity of 96% and 0.8 g/h/L, respectively [62].

Negro et al. [63] explored the utilization of inulin from stalks of Jerusalem artichoke, an alternative to the more commonly exploited JAT. There are two ways of utilizing the feedstock—simultaneous extraction and acid hydrolysis with hydrochloric acid to obtain simple sugars, followed by fermentation by S. cerevisiae, and water extraction, followed by direct fermentation by K. marxianus CECT 10875. Subjecting the raw material to 0.05 N HCl and boiling it for 10 min completed extraction of the soluble sugars (free sugars, sucrose, and soluble inulin) and simultaneous hydrolysis of the inulin to fermentable simple sugars. By varying the solid-to-liquid ratio (1:6, 1:4, and 1:3) during this step, acid-extracted juices with sugar concentrations of 38.3, 58.0, and 71.8 g/L, respectively, were obtained. The juices were fermented entirely to ethanol by S. cerevisiae within 8–9 h, producing 18.0, 27.0, and 32.5 g/L ethanol at 0.49 g/g yield. On the other hand, using water extracts of Jerusalem artichoke stalks and fermenting inulin directly with K. marxianus prolonged the fermentation substantially, with maximum ethanol concentration achieved at 30 h. Inulin concentration was negligible after 15 h, showing that the microorganisms must hydrolyze the inulin before transforming it into ethanol. Nonetheless, ethanol production reached about 18.5, 26.5, and 32.0 g/L from initial inulin concentrations of 34.0, 55.0, and 72.0 g/L with 0.49 g/g yield. The direct inulin fermentation with K. marxianus achieved 96–100% conversion, while the process with the initial hydrolysis step achieved 88–92%, likely due to S. cerevisiae being preferential to glucose and showing less efficiency with fructose [64].

3.2. Butanol

Butanol is an alcohol with four carbon atoms; therefore, it occurs in five isomeric (four structural) forms. Two biologically synthesized isomers, n-butanol and isobutanol, exist in significant amounts and are termed “biobutanol” when produced that way.

The global n-butanol market is steadily growing, having increased from 4.39 million metric tons in 2015 to 5.45 million metric tons in 2022, with forecasts for growth of up to 6.92 million metric tons by 2030 [65].

Butanol has many applications, primarily as a solvent or precursor for the chemical industry (for producing polymers, plastics, and paints) and biofuel for internal combustion engines [66,67,68]. It can be made from crude oil (via petrochemical processes) and by fermentation. Many natural and engineered microorganisms produce n- and i-butanol, such as Clostridium spp. (C. acetobutylicum, C. beijerinckii, C. pasteurianum), E. coli, some Cyanobacteria species, some Bacillus spp. (B. subtilis), Saccharomyces spp. (S. cerevisiae), and Ralstonia eutropha. The third structural isomer, 2-butanol, is synthesized by a small number of bacteria—the natural Lentilactobacillus diolivorans and engineered strains of Klebsiella pneumoniae—but the highest yield so far has not exceeded a gram per liter [63]. Most natural strains do not produce n-butanol in high titer and yield either; thus, the process is hardly industrially applicable. Therefore, much scientific research has been devoted to improving the natural capabilities of the strains, with the highest efforts devoted to the most promising Clostridium spp. [69,70,71,72,73,74,75,76].

Biobutanol is a product of acetone–butanol–ethanol (ABE) fermentation carried out by Clostridium spp. [77]. Generally, simple sugars and starch ferment during ABE fermentation, which is usually costly. Inulin, after proper pretreatment, offers a viable alternative. Additionally, some Clostridium spp. contain natural inulinases, making them suitable for advanced fermentation processes such as SSF or CBP. For example, Jiang et al. isolated and characterized Clostridium sp. strain NJ4, which processed many substrates, including inulin-rich ones such as JAT. Having efficient levanase expression made the strain suitable for CBP and yielded butanol titers and productivity (13.25 g/L and 0.09 g/L/h, respectively) comparable to those when glucose is used as substrate (14.71 g/L and 0.10 g/L/h, respectively). Additionally, when the substrate was supplemented with 30 mM sodium butyrate, butanol production reached 14.35 g/L (and total ABE production of 24.75 g/L) with productivity of 0.12 g/L/h, showcasing the viability of the isolated strain for industrial production of butanol, as well as acetone and ethanol from inulin-rich feedstock [78].

Wu et al. proposed a modification of the medium instead of the much more tedious process of genetic manipulation of the microorganism responsible for the fermentation. A simulated acid hydrolysate from JAT C. acetobutylicum L7 achieved butanol production of 4.5 g/L with productivity and yield of 0.028 g/L/h and 0.048 g/g, respectively. When the medium contained only 0.001 g/L ZnSO₄ × 7 H₂O, the yield and the productivity increased substantially, reaching 12.8 g/L, 0.089 g/L/h, and 0.122 g/g, respectively. This observation highlights the extensive effect of micronutrients on the overall efficiency of biotechnological processes [79].

Ujor et al. utilized inulin from two different sources (roots from chicory and Kazakh dandelion (Taraxacum kok-saghyz)) for butanol production by C. saccharobutylicum P262 via the ABE process without prior hydrolysis. Following optimization of the water extraction procedure (90 °C, 30 min, 0.1% Na₂CO₃, 1: 6 (w/v) solid to solvent ratio) of inulin from the raw biomass, the authors obtained 41.8 g/L inulin, and 35.2 g/L soluble sugars extracted from the chicory roots, as well as 36.0 g/L inulin and 33.0 g/L soluble sugars from the TKS roots. With TKS root extract used as a substrate, C. saccharobutylicum P262 produced 4.88 g/L butanol, utilizing 25.2 g of the available 44.1 g total sugars (with total ABE production, yield, and productivity of 8.5 g/L, 0.33 g/g and 0.12 g/L/h, respectively). Chicory root extract as a substrate gave 8.58 g/L butanol, consuming 40.5 g of the 77.0 g available sugars (with ABE production, yield, and productivity of 12.5 g/L, 0.32 g/g, and 0.21 g/L/h, respectively) [80].

In two studies by Sarchami and Rehmann, the authors employed advanced optimization techniques (central composite design and response surface methodology) to optimize the enzymatic [81] and acid hydrolysis [82] of JAT to maximize fermentable sugar yield and utilize them in ABE fermentation. Following Bekers et al., water extraction was performed on JAT in the first study [83]. The substrate concentration, pH, and temperature were optimized, with inulin conversion percent as a response. The results showed the optimal conditions for hydrolysis as enzyme loading of 10 U/g, substrate concentration of 60 g/L, pH 4.8, and T 48 °C for 24 h, yielding inulin conversion of 94.1% and 55 g/L total sugars (41 g/L glucose and 14 g/L glucose). Using the enzymatic hydrolysate as a substrate for 60 h, ABE fermentation by C. saccharobutylicum DSM 13864 produced 9.6 g/L butanol with a yield of 0.33 g/g, utilizing 45.6 g/L of the available sugars [81]. In their second study, optimization of the technological parameters (temperature, pH (acid concentration), hydrolysis time, and mineral acid used (HCl, H₂SO₄, and H₃PO₄)) showed that H₂SO₄ hydrolyzes 98.5% of the available inulin for 30 min at 97 °C and pH 2.0. Finally, a 60 h ABE fermentation with C. saccharobutylicum DSM 13864 produced 9.8 g/L butanol and overall ABE production of 15.1 g/L with productivity of 0.25 g/L/h, utilizing 48.2 g/L of the available sugars, which corresponds to a yield of 0.31 g/g, 80% of the theoretical maximum yield [83].

Chen et al. (

Table 3. Butanol produced from inulin sources.

Inulin Source	Pretreatment	Fermentation Mode	Species	Butanol (g/L)	Conversion (%)	Yield (g/g)	Productivity (g/L/h)	Reference
JAT	Enzymatic	CBP 1	Clostridium sp. NJ4	13.25	ND 2	ND	0.09	[78]
JAT 3	Enzymatic	CBP 1	Clostridium sp. NJ4	14.4 (24.6) 4	ND	ND	ND	[78]
JAT	Acid	Batch	C. acetobutylicum L7	4.5	ND	0.048	0.028	[79]
JAT 5	Acid	Batch	C. acetobutylicum L7	12.8	ND	0.122	0.089	[79]
JAT	Steam and pressure	Batch	C. saccharobutylicum P262	8.6 (12.5) 4	ND	0.32	0.21	[80]
Chicory roots 6	Steam and pressure	Batch	C. saccharobutylicum P262	4.88 (8.5) 4	ND	0.33 4	0.12	[80]
JAT	Enzymatic	Batch	C. saccharobutylicum DSM 13864	9.6	ND	0.33 4	ND	[81]
JAT	Acid (H2SO4)	Batch	C. saccharobutylicum DSM 13864	9.8 (15.1) 3	80.0	0.31	0.25	[81]
JA juice 7	Acid	Batch	C. acetobutylicum L7	8.67	93.6	0.192	ND	[84]
JA juice 7	Acid	Batch	C. acetobutylicum L7	11.21	94.8		ND	[84]

¹ CBP, Consolidated bioprocessing; ² ND, no data; ³ In medium supplemented with sodium butyrate; ⁴ Value of total ABE fermentation products; ⁵ Zn-supplemented medium; ⁶ Combined with Taraxacum kok-saghyz roots; ⁷ Sugar-supplemented medium.

) utilized acid hydrolysis of Jerusalem artichoke juice for ABE batch fermentation by C. acetobutylicum L7. By using 93.6% of the available 48.36 g/L, the microorganism produced 8.67 g/L butanol with a yield of 0.192 g/g at the end of a 60 h batch fermentation, with a ratio of butanol:acetone:ethanol = 0.58:0.36:0.06. By artificially increasing the total sugar content to 62.87 g/L, the equilibrium in the ABE process was shifted more towards butanol, changing the ratio of B:A:E = 0.64:0.29:0.05, reaching 11.21 g/L butanol production and utilizing 94.8% of the available sugars, demonstrating a relatively easy and cheap method to boost both the production of one particular product over the rest and boosting overall efficiency slightly. An additional increase in total initial sugars had a detrimental effect, drastically increasing the residual sugars after the fermentation, demonstrating that this sugar concentration is the limit of the particular strain employed [84].

Prospects for butanol production focus on increasing process yield, productivity, and overall efficiency. Utilizing renewable, non-edible carbohydrate sources such as inulin-rich biomass helps reduce reliance on fossil fuels and carbon emissions. Butanol production and the ABE fermentation process are great examples of the biorefinery concept, which produces multiple added-value products from an underused feedstock, further promoting the principles of a circular economy.

3.3. 2,3-Butanediol (2,3-BD)

2,3-Butanediol (2,3-BD) exists in three isomeric forms, two of which are optically active, L(+)-2,3-BD (2S, 3S) and D(−)-2,3-BD (2R, 3R), and meso-2,3-BD (2R, 3S or 2S, 3R), which is optically inactive. These isomers can have different physical and chemical properties, essential for their use in various industrial applications [85]. Different microorganisms produce different stereoisomers; many bacterial strains form a mixture of stereoisomers during fermentation [86]. At room temperature, 2,3-BD is a colorless, odorless liquid and can also form crystals under specific conditions, depending on the stereoisomer. It is soluble in water, alcohols, ethers, and ketones, making it a versatile solvent in various chemical processes. Its high boiling point and low freezing point make it useful in applications where thermal stability over a wide range of temperatures is essential. These physical properties contribute to its many applications, such as an anti-freeze agent in pharmaceuticals, as an industrial solvent, and its use in the production of biofuels [87].

The global market for 2,3-BD will grow significantly, with projections estimating it could reach $300 million by 2030 [88]. As a result, biorefining biomass to produce 2,3-BD is sustainable, as renewable biomass offers a promising pathway toward achieving a carbon-neutral economy as an environmentally friendly alternative to traditional chemical production methods [89,90]. The wide range of applications of 2,3-BD motivates further research on the biotechnological production process for its synthesis as an alternative to traditional chemical methods [91].

Microbial production of 2,3-BD has a history of more than 100 years. In 1906, Harden and Walpole were the first to study the microbial production of 2,3-BD using Klebsiella pneumoniae. Following this discovery, Donker (1926) continued researching the microbial production of 2,3-BD using Paenibacillus polymyxa. Fulmer et al. (1933) were the first to propose the industrial production of 2,3-BD, recognizing its potential applications in various industries [86].

Several wild-type bacterial strains are known to produce 2,3-BD, including K. pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes, and Serratia marcescens. Although they produce high titers of 2,3-BD, these strains belong to class 2 (pathogenic) microorganisms [92]. Bacillus sp. has Generally Regarded as Safe (GRAS) status, which minimizes safety concerns, particularly for food, pharmaceutical, and biotechnological applications [93]. Also, well-developed and optimized fermentation techniques are available for their industrial-scale cultivation. However, not all these hosts naturally possess the metabolic pathways to produce 2,3-BD from inulin [2,91]. Therefore, in addition to native 2,3-BD producers, engineered hosts such as E. coli, S. cerevisiae, B. licheniformis, and Paenibacillus sp. were developed. As a result, engineered strains have achieved titers of 2,3-BD comparable to those of the native microbial producer [94].

The feedstock used in fermentation often constitutes the largest share of production expenses, including in producing compounds like 2,3-BD, and there has been a shift toward renewable, non-food, and cheaper sources to use as substrates in the last two decades. Gao et al. [95] conducted one-step fermentation of raw inulin extract from JAT by P. polymyxa ZJ-9 to produce (R,R)-2,3-BD. The Plackett–Burman design application evaluated inulin, K₂HPO₄, and NH₄Cl as key components influencing fermentation. Under the optimized conditions, P. polymyxa ZJ-9 gained 36.92 g/L R,R-2,3-BD with over 98% optical purity and 0.88 g/g productivity. The raw inulin extract provided the highest yield among the different carbon sources tested. This one-step process eliminates the need for inulin hydrolysis, reducing raw material costs and making the process more practical and cost-effective for industrial applications. In their study, Li et al. [96] used levanase with high inulinase activity and stability at high pH and temperature, identified from B. licheniformis ATCC 14580. This strain efficiently produced 2,3-BD from fructose at 50 °C during SSF with levanase hydrolyzing inulin. The fed-batch SSF process produced 103.0 g/L of 2,3-BD in 30 h, with a high productivity of 3.4 g/L/h. This finding suggests that this thermophilic strain and the developed SSF process offer a promising alternative for efficient 2,3-BD production from inulin [90].

Park et al. [97] utilized JAT as a feedstock to produce 2,3-BD. The authors showed that the critical enzyme with high inulinase activity was the potential fructan-hydrolyzing SacC. The responsible sacC gene was expressed in E. coli, followed by back-introduction of sacC into Bacillus sp. BRC1, which increased the enzymatic activity more than twofold. As a result, 2,3-BD production from JAT rose from 3.98 g/L to 8.10 g/L. In fed-batch fermentation, the recombinant strain achieved a 2,3-BD maximum output of 28.6 g/L, with a high theoretical yield of 92.3% [98] (

Table 4. Production of 2,3-butanediol by natural and engineered strains from inulin-containing feedstocks.

Inulin Source	Pretreatment	Fermentation Mode	Species/Strain	2,3-BD (g/L)	Yield (g/g)	Productivity (g/L/h)	Reference
Frutafit® CLR	No	SSF	B. licheniformis ΔsacB	128.7	0.429	1.65	[91]
JAT	No	Batch	P. polymyxa ZJ-9	36.92	ND	0.88	[95]
JAT	Enzymatic	Fed-batch SSF	B. licheniformis ATCC 14580	103.0	ND	3.4	[96]
Frutafit® HD	No	SSF	B. licheniformis T26	18.5	0.1	ND	[97]
JAT	Enzymatic	Fed-batch	Bacillus sp. BRC1	28.6	ND	ND	[98]
JAT	Enzymatic	SSF, FB	K. pneumoniae	81.58	ND	ND	[99]
JAT	Enzymatic	Fed-batch SSF	K. pneumoniae	91.63	ND	ND	[99]
JAT	Acid	Batch	K. pneumoniae	80.5 *	ND	ND	[100]
JAT stalks	No	Batch	K. pneumoniae H3	80.4 *	0.426 *	2.23 *	[101]

* Value of butanol + acetoin products; FB, fed-batch; ND, not determined.

).

Tsigoriyna et al. [97] highlighted the potential for enhancing 2,3-BD production by B. licheniformis 24 by expressing a heterologous inulinase inu gene from Lacticaseibacillus paracasei DSM 23505, which encodes fructan-β-fructosidase (EC 3.2.1.80). Two variations of the gene—a full-length version (3.6 kb with Big3 cell wall attachment domains) and a truncated version (2.2 kb, lacking these domains)—were successfully cloned into E. coli and B. licheniformis 24. The full-length inulinase with full-length inu variant (T26) showed eightfold higher inulinase activity than the wild type. The truncated version (T14) exhibited mostly extracellular activity but was weaker, highlighting the importance of the Big3 cell wall attachment domains for enzyme functionality. During fermentation with chicory flour Frutafit^® HD, T26 produced significant amounts of 2,3-BD and acetoin, while T14 and the wild strain only utilized mono- and disaccharides. The recombinant T26 utilized about 140 g/L of insoluble inulin but was limited in 2,3-BD synthesis by the accumulation of unconverted sucrose, resulting in moderate product yields of 18.5 g/L 2,3-BD and 8.2 g/L acetoin [97]. While this engineered strain showed promise, further genetic improvement of B. licheniformis 24 by inactivating the sacB gene for levansucrase achieved the highest concentrations for inulin-derived 2,3-BD. By batch fermentation with soluble inulin Frutafit^® CLR, the mutant BLΔsacB produced significantly fewer exopolysaccharides than the wild type, allowing pH maintenance at values favoring 2,3-BD synthesis. At pH 6.50, BLΔsacB reached a record titer of 128.7 g/L 2,3-BD, with productivity of 1.65 g/L/h and a yield of 85.8% of the theoretical maximum [91].

Although opportunistic pathogens, K. pneumoniae, K. oxytoca, and S. marcescens remained among the most efficient producers of 2,3-BD. Sun et al. successfully developed a method for producing 2,3-BD from JAT using K. pneumoniae, exploring SHF and SSF approaches. In these processes, the pretreated tubers were hydrolyzed to glucose and fructose using exogenous inulinase. The concentration of 2,3-BD reached 81.59 g/L in batch SSF and 91.63 g/L in fed-batch SSF after 40 h. The SSF process resulted in a 30.3% higher product concentration and 83.2% greater productivity than fed-batch SHF. These findings highlight that JAT is a suitable substrate for 2,3-BD production and that fed-batch SSF is more cost-effective [99]. Li et al. developed a fermentation process using the stalk and tuber as feedstock to utilize the entire Jerusalem artichoke plant. After adding tuber to the stalk hydrolysate to increase sugar concentration, the sugars were converted into 2,3-BD and acetoin by K. pneumoniae. In fed-batch SSF and a stage-shift aeration strategy, 80.5 g/L of 2,3-BD plus acetoin were produced in 68 h. This method increased the concentration, yield, and productivity by 16.9%, 16.8%, and 23.4%, respectively, compared to SSF with constant aeration, demonstrating the effectiveness of adding tubers to enhance 2,3-BD production through fermentation [100]. In their study, Dai et al. also used K. pneumoniae strain H3, which can convert inulin without needing external inulinase, but 2,3-BD production was below 50 g/L. The enzyme used was an intracellular inulinase with an optimal pH range of 6–7 and temperature of 30 °C. The strain’s ability to utilize inulin depended on DP. Hence, an acidic pretreatment of inulin included adjusting the medium’s pH to 3.0 and autoclaving. Under optimized medium and fermentation conditions, batch fermentation yielded 80.4 g/L 2,3-BD and acetoin, with a productivity of 2.23 g/L/h and a yield of 0.426 g/g from a 202.6 g/L initial sugar concentration [101].

4. Organic Acid Production from Inulin

4.1. Lactic Acid

Lactic acid (LA) is a naturally occurring organic acid first discovered in sour milk by Carl Wilhelm Scheele in 1780. It is GRAS-classified, therefore, harmless for food and pharmaceutical use. Louis Pasteur pioneered the microbial fermentation of LA in 1857. Later, in 1881, Fermi successfully extracted lactic acid, which marked a significant step in enabling industrial production [102]. LA is of significant biotechnological importance due to its wide range of applications across various industries—in textiles, cosmetics (as an exfoliant and moisturizer), the medical and pharmaceutical sectors (for drug formulations, biodegradable sutures, and controlled drug delivery systems), and as a precursor for producing biodegradable plastics like polylactic acid (PLA), to name just a few. LA is also widely used in the food industry for several purposes, such as preserving food by lowering pH (acidifier), preventing microbial growth (antibacterial), and enhancing flavors [102]. LA exists in two enantiomeric forms/isomers: L(+)-lactic acid and D(-)-lactic acid. The L(+)-form is the naturally occurring form in many biological systems, while the D(-)-form can be produced by certain bacteria and through chemical synthetic processes. Both forms have different applications. The specific form produced depends on the microorganism or production process employed. Some production routes lead to a racemic mixture containing both L(+) and D(-) forms [103]. Historically, LA production has relied on refined sugars from grain crops (such as glucose or sucrose) to achieve high yields. This dependency on crop-based sugars creates concerns regarding food safety, as these crops are also used for human consumption, impacting global food security.

In the past decade, there has been significant interest in producing LA using inulin-type feedstocks. As early as 1942, researchers Andersen and Greaves explored using JAT powder as a substrate for producing D-lactic acid [104]. Cai et al. obtained 144.08 g/L LA, with a yield of 0.67 g/g, and an average productivity of 4.37 g/L/h during a fermentation process from 215 g/L inulin-rich JAT, which was directly converted into LA without the addition of an external nitrogen source under non-sterile conditions using the strain L. paracasei. This process was successfully scaled up and conducted in bioreactors from 5 L to 50 L. The strain L. paracasei NJ reached viable cell counts of 142.3–301.5 × 10⁸ CFU/mL, indicating that the fermentation products could also serve as promising probiotics for food additives. Transcriptional analysis revealed that the strain utilized inulin through extracellular β-fructosidase and fructose transporters encoded by the fosRABCDXE operon. In response to nitrogen limitation, the genes related to amino acid biosynthesis and peptide/amino acid ABC transport systems were upregulated, enabling the strain to thrive without external nitrogen supplementation. This study shows the feasibility of using Jerusalem artichoke as a raw material for high-yield LA production. It provides insights into the genetic regulation of inulin metabolism in L. paracasei NJ. This process can produce valuable food additives and probiotics while offering a new perspective on inulin metabolism [105]. Choi et al. also reported that L. paracasei strains can efficiently ferment FOS with a DP of up to 13 from Jerusalem artichoke without requiring inulin pretreatment. Furthermore, these strains could also metabolize inulin with a higher DP, like that found in dahlia. Six strains of L. paracasei (KCTC3165, 3166, 3169, 3510, 13090, and 13169) and L. casei KCTC3109 were tested. Initially, lactic acid production was inefficient when cultivated in hot-water extract of JAT without supplementation. However, when the extract was supplemented with 1/5 strength of MRS, most L. paracasei strains, except for KCTC3166, produced LA efficiently. The KCTC13090 and KCTC13169 strains demonstrated significantly higher LA production than other L. paracasei strains. In contrast, the L. casei strain showed much lower LA production. This aligns with previous findings, which indicated that efficient lactic fermentation with L. casei required acid pretreatment of the JAT to hydrolyze the inulin. L. paracasei KCTC 13169 was identified as the most efficient inulin-fermenting strain, producing approximately six times more LA than the L. casei strain when fermenting Jerusalem artichoke without pretreatment. Using L. paracasei KCTC13169 in a 5 L fermenter with 111.6 g/L of sugar and 5 g/L of yeast extract resulted in 92.5 g/L of LA after 72 h fermentation, with 16.8 g/L of fructose remaining with 98% conversion efficiency [106] (Table 5).

Ge et al., for the first time, used a strategy for the direct production of L-lactic acid from JAT by applying co-fermentation with Aspergillus niger SL-09 and Lactobacillus sp. G-02 in a 7 L fermenter. This strategy significantly boosted the production of inulinase and invertase by A. niger SL-09 when Lactobacillus sp. G-02 was introduced after 12 h of cultivation, reaching 275.6 and 571.8 U/mL in 60 h—over five times higher than the single-strain culture. During the subsequent SSF, the highest concentration of L-lactic acid, 120.5 g/L, was achieved in 36 h of fed-batch fermentation, with a high conversion efficiency of 94.5% [107]. To further enhance lactic acid productivity, Ge et al. studied the effect of the addition of sodium citrate. By performing SSF at 40 °C after one h of hydrolysis and adding 10 g/L of sodium citrate, an L-lactic acid concentration of 141.5 g/L was achieved in 30 h with a productivity of 4.7 g/L/h. The conversion efficiency was 93.6% of the theoretical yield, and the LA yield was 52.4 g per 100 g of Jerusalem artichoke flour. This high concentration and productivity make L. casei G-02 a promising candidate for large-scale commercial L-lactic acid production from JAT [108]. In addition to JAT [109,110], a promising inulin source for LA production is chicory flour with lower DP, which is amenable in large-scale processes due to its solubility [111,112].

Table 5. Production of lactic acid from inulin-containing sources.

Inulin Source	Hydrolysis Type	Fermentation Mode	Strain	LA (g/L)	Conversion (%)	Yield (g/g)	Productivity (g/L/h)	Reference
JAT	Enzymatic	Batch	L. paracasei NJ	144.08	ND	0.67	4.37	[105]
JAT	Enzymatic	SSF, FB	L. paracasei KCTC 13169	92.5	98.0	ND	ND	[106]
JAT	Enzymatic	SSF	A. niger SL-09, Lactobacillus sp. G-02	120.5	94.5	ND	ND	[107]
JAT	Enzymatic	SSF	A. niger SL-09, Lactobacillus sp. G-02	141.5	93.6	0.524	4.7	[108]
JAT	Enzymatic	Fed-batch	B. coagulans XZL4	134.0	ND	0.96	2.5	[109]
JAT	Enzymatic	-	L. lactis	142.0	-	ND	ND	[110]
Chicory flour	Enzymatic	Batch	L. paracasei DSM 23505	123.7	91.0	ND	ND	[113]
Chicory	Enzymatic	SSF	L. bulgaricus CGMCC 1.6970	123.6	97.9	ND	ND	[114]

Table 5. Production of lactic acid from inulin-containing sources.

Inulin Source	Hydrolysis Type	Fermentation Mode	Strain	LA (g/L)	Conversion (%)	Yield (g/g)	Productivity (g/L/h)	Reference
JAT	Enzymatic	Batch	L. paracasei NJ	144.08	ND	0.67	4.37	[105]
JAT	Enzymatic	SSF, FB	L. paracasei KCTC 13169	92.5	98.0	ND	ND	[106]
JAT	Enzymatic	SSF	A. niger SL-09, Lactobacillus sp. G-02	120.5	94.5	ND	ND	[107]
JAT	Enzymatic	SSF	A. niger SL-09, Lactobacillus sp. G-02	141.5	93.6	0.524	4.7	[108]
JAT	Enzymatic	Fed-batch	B. coagulans XZL4	134.0	ND	0.96	2.5	[109]
JAT	Enzymatic	-	L. lactis	142.0	-	ND	ND	[110]
Chicory flour	Enzymatic	Batch	L. paracasei DSM 23505	123.7	91.0	ND	ND	[113]
Chicory	Enzymatic	SSF	L. bulgaricus CGMCC 1.6970	123.6	97.9	ND	ND	[114]

Petrova et al. [113] developed highly efficient SSF using chicory flour and the strain L paracasei DSM 23505. In batch fermentation, with optimized medium and conditions, 136 g/L of chicory flour (composed of 89.3% inulin and 10.7% of a mixture of sucrose, fructose, and glucose) was fully converted into 123.7 g/L of LA with a conversion rate of 91%. The high efficiency, low-cost medium, and simple process highlight the potential of chicory flour in industrial LA production.

In another study, Xu et al. obtained 123.6 g/L D-lactic acid, yielding 97.9% from 120 g/L chicory-derived inulin by L. bulgaricus CGMCC 1.6970. SSF showed superior results for D-lactic acid production than the SHF process, as SSF avoided fructose inhibition and ensured complete inulin hydrolysis. Additionally, SSF provided another benefit: higher optical purity of D-lactic acid (>99.9%) and fewer residual sugars. This approach demonstrates a highly efficient method for producing D-lactic acid from non-food grain sources [114].

The thermophilic Bacillus strain’s potential for industrial applications lies in its impressive abilities for lactate production, minimal nutritional requirements, suitability for fermentation without sterilization, and ease of maintaining stock cultures. Wang et al. used the thermophilic Bacillus coagulans XZL4 for the efficient production of high-purity L-lactate from 267 g/L JAT powder which was hydrolyzed with inulinase to 140 g/L of total reducing sugars (fructose and glucose). In the fed-batch fermentation process, 134 g/L of L-lactate with an average productivity of 2.5 g/L/h and a yield of 0.96 g/g of reducing sugars were achieved. The final product had an optical purity of 99%. This approach offers a cost-effective and promising method for producing L-lactate [109].

Shi et al. developed a novel method for producing L-lactic acid using immobilized cells. They employed a fibrous-bed bioreactor to immobilize Lactococcus lactis cells to ferment Jerusalem artichoke hydrolysates. This process achieved 142 g/L L-lactic acid concentration during fed-batch fermentation [110].

Producing lactic acid from inulin is a promising research and industrial development area. Optimizing fermentation processes can create sustainable, high-yield production methods to meet growing demand, particularly for biodegradable materials and food additives.

4.2. Citric Acid

The production of citric acid (CA), a key intermediate in the Krebs cycle, is one of the oldest and most well-established technologies for organic acid production on an industrial scale. Like LA, CA has GRAS status, meaning it is safe for food and other consumer products. During the late 19th and early 20th centuries, commercial production of CA was carried out by extraction from lemons, first performed around 1826 in England using imported Italian lemons. Due to increasing demand and the limitations of lemon-based extraction, industrial production shifted to biosynthesis in 1917. James Currie discovered that the fungus A. niger could produce citric acid via fermentation. This process is much more efficient and cost-effective than extraction from citrus fruits, and allows citric acid to be made from cheaper sources like molasses or sucrose, gradually replacing the lemon-based extraction process [115,116].

The most commonly used species for industrial production of CA to date remains A. niger due to its high efficiency and yield. Other citric acid-producing fungi include various species from the genus Aspergillus—A. awamori, A. flavus, A. nidulans, A. phoenicus, and A. luchuensis—as well as fungi from other genera like Acremonium sp., Absidia sp., Botrytis sp., Eupenicillium sp., Penicillium restrictum and Penicillium janthinellum, Talaromyces sp., Mucor piriformis, and Trichoderma viride [117].

CA is one of the most economically feasible products of microbial production since it has enormous applications in various industries, including food, beverages, pharmaceuticals, cosmetics, and chemicals. It is used as a preservative and flavor enhancer in the food and beverage industries, as a stabilizer for vegetable oils and fats in food processing, and as a complexing agent and bleaching component in washing detergents and cleaning products. CA can help balance acidity and prevent oxidative deterioration of flavors and colors in food [118].

The first use of inulin as a substrate for CA was reported by Drysdale et al. in 1995 [119]. They engaged A. niger ATCC 9142 to produce 14 g/L CA by surface fermentation. However, the study demonstrated that inulin yields were lower than sucrose’s. By modifying the fermentation process, where air was passed over the surface of the medium, CA yields improved substantially, reaching 29 g/L after 24 days of incubation. Then, Lotfy et al. conducted a study on an optimization approach based on statistical designs to improve CA production in submerged cultures. A two-level Plackett–Burman design was initially applied to screen the fermentation medium components, significantly affecting CA production. This method achieved a near-optimal medium formulation, increasing CA yield fivefold. Subsequently, response surface methodology (RSM) was employed to determine the best process conditions. The optimal medium contained pretreated beet molasses at 240.1 g/L, corn steep liquor at 10.5 g/L, and a spore concentration of 1 × 10⁸ spores/mL. This formulation resulted in an optimal CA yield of 87.81%, 14 times higher than the yield from the basal medium. Furthermore, a five-level central composite design was used to determine the optimal values for fermentation factors. The estimated optimum conditions were an initial pH of 4.0, an aeration rate of 6500 mL/min, and a fermentation temperature of 31.5 °C [120]. Modern challenges in CA production include achieving high titers, productivity, and yield while adhering to cleaner production principles, energy savings, and sustainable development. Rakicka et al. used genetically engineered Yarrowia lipolytica strains to produce CA from inulin. The genetic modification involved expressing the inu1 gene encoding inulinase from K. marxianus CBS6432, which enabled the Y. lipolytica strains to hydrolyze inulin into simple sugars. A repeated batch culture strategy achieved the highest possible citric acid yield. The Y. lipolytica AWG7 INU 8 strain produced over 200 g/L of CA with a productivity of 0.51 g/L/h and a yield of 0.85 g/g. This result is one of the highest values reported from Yarrowia yeast [121].

Another study investigated the applicability of inulin and glycerol as co-substrates for the efficient production of erythritol and CA by newly engineered strains of Y. lipolytica. The engineered strain, Y. lipolytica Wratislavia K1, was modified to express the mentioned gene inu1 of K. marxianus. The process was carried out in two stages: first, inulin was used for biomass formation, followed by adding glycerol to initiate erythritol biosynthesis. The engineered strain K1 INU 6 produced the highest titer of erythritol: 120.9 g/L with a yield of 0.6 g/g. In fed-batch culture, the strain produced 105.2 g/L CA from 200 g/L inulin after 235 h. The maximum activity of inulinase during this fermentation reached 14,000 U/g of cell dry mass. This study highlights the potential of new Y. lipolytica transformants for producing erythritol and CA from inexpensive raw materials like inulin and glycerol, making the process economically viable [122]. One of the disadvantages of using wild-type Y. lipolytica strains for commercial CA production is the simultaneous secretion of iso-citric acid (ICA). ICA contaminations greater than 5% disturb the crystallization of CA during the purification process, which can reduce the efficiency of CA production. The ratio of CA to ICA produced by Y. lipolytica depends on the substrate and strain used. Wild-type strains tend to secrete 10–12% ICA when grown on carbohydrates or glycerol and even higher proportions (35–45% ICA) when grown on plant oils or n-alkanes. Modifying cultivation conditions (temperature, pH, air saturation, and iron concentration) can influence the CA/ICA ratio. Additives like acetate or fluoroacetate also impact the production ratio. Therefore, Y. lipolytica strains are challenging and need optimization of CA production to minimize unwanted ICA contamination and genetic and process engineering [123].

Y. lipolytica SWJ-1b, isolated from the gut of a marine fish in the Bohai Sea, produced a large amount of CA. However, this strain cannot synthesize and secrete exo-inulinase. Inulinase gene inu1 from K. marxianus CBS 6556 was cloned into a surface display vector, which was then transformed into a uracil mutant of Y. lipolytica SWJ-1b. The expressed inulinase hydrolyzed inulin directly, allowing the engineered yeast to produce CA from inulin. Displayed inulinase activity was measured at 22.6 U/mg of cell dry weight after 96 h of growth. At flask fermentation, the recombinant produced 77.9 g/L CA and 5.3 g/L ICA acid from inulin. In a 2 L fermentation system, 68.9 g/L CA and 4.1 g/L ICA were obtained after 312 h. Thus, a successful genetic engineering approach enabled Y. lipolytica to hydrolyze inulin and produce CA efficiently [124] (

Table 6. Production of citric acid from inulin-containing feedstocks.

Inulin Source	Fermentation Type	Species/Strain	Yield (g/L)	Reference
Chicory flour	Surface fermentation	A. niger ATCC 9142	14	[113]
Chicory flour	Air-passed surface fermentation	A. niger ATCC 9142	29	[113]
JAT	Repeated-batch	Y. lipolytica AWG7 INU 8	200	[115]
Inulin and glycerol	Fed-batch	Y. lipolytica Wratislavia K1 INU 6	105.2	[116]
JAT	Flask-batch	Y. lipolytica SWJ-1b	77.9 (5.3) *	[118]
JAT	2 L fermentation system	Y. lipolytica SWJ-1b	68.9 (4.1) *	[118]

* Value of ICA.

).

In conclusion, CA production from inulin is promising due to the abundance of inulin-rich crops and the growing interest in sustainable and cost-effective bioprocesses. It holds great potential for sustainability and cost-effectiveness. Future research will likely focus on improving microbial strains, optimizing fermentation processes, utilizing inulin-rich waste, and scaling production. As part of the broader trend toward bio-based chemicals, CA production from inulin could become essential to circular economy models.

4.3. Poly-γ-glutamic Acid (γ-PGA)

Microbial biopolymers are gaining global popularity due to their eco-friendly, degradable, and non-toxic nature compared to synthetic, non-degradable polymers. Poly-γ-glutamic acid (γ-PGA) is a specific example as it is expensive because producing even small quantities can cost relatively large amounts, and the production process involves complex biological and chemical synthesis. This naturally occurring anionic homo-polyamide polymer is unique in its structure, composed of D- and L-glutamic acid units connected via γ-amide linkages between α-amino and γ-carboxylic acid groups [125].

γ-PGA is recognized for its favorable characteristics, making it a valuable biopolymer in various applications. It is an entirely biodegradable polymer, which means it can be broken down by microorganisms into non-toxic byproducts, contributing to environmental sustainability. It is water-soluble, making it easily used in various industries. It is edible and suitable for food and pharmaceutical applications. Its safety for human consumption is advantageous for potential food packaging and additives applications. It is non-immunogenic, meaning it does not provoke an immune response in humans, which is particularly important for biomedical applications, such as drug delivery systems and tissue engineering [126].

Ivánovics et al., at the start of the 20th century, discovered γ-PGA as a component of the capsule of B. anthracis, the bacterium responsible for anthrax as released into the medium after cell autolysis, aging, or autoclaving. Researchers discovered that γ-PGA forms part of the protective capsule surrounding B. anthracis, contributing to the bacterium’s virulence by helping it evade the host’s immune system. Further studies have demonstrated the presence of γ-PGA in the extracellular viscous material produced by several non-pathogenic Bacillus species, such as B. subtilis, B. licheniformis, B. megaterium, and B. halodurans [127].

Gram-positive bacteria, some archaea, and certain eukaryotic organisms synthesize γ-PGA. Some archaeal species, particularly those in extreme environments, have been identified as γ-PGA producers. The roles of γ-PGA in bacterial physiology are in two distinct contexts: anchored PGA and released PGA. Anchored PGA, as a virulence factor in pathogenic bacteria, forms a protective capsule that serves as a virulence factor. Released PGA is excreted into the surrounding environment to help protect the bacterium from environmental stressors, such as desiccation, UV radiation, or other unfavorable conditions, thereby enhancing survival [128].

Various strains capable of producing γ-PGA have been identified, with Bacillus species, particularly B. subtilis and B. licheniformis, being the most effective producers [129,130]. Shih et al. classified γ-PGA-producing bacteria into two categories based on their nutrient needs for synthesis. One category requires L-glutamic acid in the growth medium, while the other does not. L-glutamate-independent γ-PGA producers are preferred for industrial production due to their simpler fermentation processes and lower costs than L-glutamate-dependent producers. Nonetheless, their reduced productivity limits their industrial use [131,132].

The formulation of media for producing γ-PGA remains a significant industrial challenge, primarily due to the associated costs. High costs can arise from the need for specific nutrients, such as L-glutamic acid. The glutamic acid monomers incorporated into γ-PGA can be sourced from the culture medium used for microbial fermentation or from precursors derived from the tricarboxylic acid (TCA) cycle by microorganisms [131,133].

Qiu et al. identified and characterized the key enzyme responsible for inulin metabolism. An inulin hydrolase CscA was identified and isolated. This novel enzyme exhibited relatively high activity and stability in alkaline pH compared to other inulin hydrolases, making it a promising candidate for hydrolysis in biorefinery processes. The overexpression of the CscA gene significantly increased inulin consumption, leading to a γ-PGA concentration of 18.95 g/L, representing a 19.2% increase, with a productivity of 0.29 g/L/h [134]. The work demonstrates a sustainable approach to efficiently producing γ-PGA from renewable resources through metabolic engineering (

Table 7. γ-PGA produced from inulin sources.

Inulin Source	Fermentation Mode	Species/Strain	Yield (g/L)	Productivity (g/L/h)	Reference
JAT	Batch	B. amyloliquefaciens NB	18.95	0.29	[134]
JAT	Fed-batch	B. amyloliquefaciens NB	32.14	ND	[135]
JAT	Batch	B. amyloliquefaciens NX-2S	6.85	ND	[136]
JAT	Batch	B. amyloliquefaciens NX-2S	39.4	0.43	[136]
JAT	Fed-batch	B. amyloliquefaciens NX-2S154	14.83	ND	[137]
JAT	Batch	B. amyloliquefaciens NB	17.62 1	ND	[138]
JAT	SSF + inulinase (40 IU/g inulin)	B. amyloliquefaciens NX-2S154	18.54	ND	[139]
JAT	Repeated batch	B. amyloliquefaciens NX-2S154	19.93	0.28	[139]

¹ Value of LMW-γ-PGA; ND, not determined.

).

In another study, Qiu et al. [134] used wild-strain B. amyloliquefaciens NB, capable of producing γ-PGA without relying on glutamate but utilizing inulin-containing feedstock. The authors employed CRISPR-Cas9n to improve γ-PGA production by modifying the strain to optimize inulin hydrolysis, sugar metabolism, and γ-PGA synthesis. By maximizing the overexpression of native inulin hydrolase, along with levanase and endo-inulinase, the system achieved a significant boost in both extracellular inulinase activity, reaching 22.63 U/mL, and γ-PGA production, which reached 18.92 g/L. This enhancement significantly improved the efficiency of inulin breakdown and the subsequent synthesis of γ-PGA. The system improved sugar metabolism by blocking specific biosynthetic pathways and deleting the γ-PGA hydrolase gene cwlO. This resulted in a maximum γ-PGA production of 32.14 g/L after 120 h of fed-batch fermentation [135]. Qiu et al. aimed to develop a cost-effective, non-food fermentation process for producing γ-PGA using a novel strain of B. amyloliquefaciens NX-2S. The strain efficiently assimilated inulin from JAT without hydrolytic treatment, outperforming other carbohydrates. A transcriptomic study revealed upregulation of the γ-PGA synthetase genes (pgsB, pgsC, pgsA), the regulatory genes (comA, degQ, degS), and the glutamic acid biosynthesis gene (glnA) in the presence of inulin. Even without the addition of external glutamate, the NX-2S strain produced 6.85 ± 0.22 g/L γ-PGA during fermentation. When exogenous glutamate was added, the yield significantly increased to 39.4 ± 0.38 g/L, with a productivity of 0.43 ± 0.05 g/L/h in batch fermentation. This study presents a promising method for producing high-value products through non-food fermentation [136].

To further improve and explore the use of Jerusalem artichoke in γ-PGA production, a B. amyloliquefaciens NX-2S154 strain was developed through atmospheric and room temperature plasma mutagenesis. This strain produced 14.83 g/L γ-PGA in batch fermentation using raw inulin extract. The study further investigated SSF by adding commercial inulinase of 40 IU/g inulin for γ-PGA production. The process improved the utilization of inulin, avoided substrate inhibition, and gained up to 18.54 g/L γ-PGA. Column immobilization of inulinase costs and repeated batch cultures decreased cost and demonstrated higher stability and simplicity, reaching a maximum γ-PGA concentration of 19.93 ± 0.38 g/L and a productivity of 0.28 ± 0.011 g/L h. This study presents an efficient method for γ-PGA production using Jerusalem artichoke as a feedstock [137].

Sha et al. conducted a study on synthesizing low-molecular-weight poly-γ-glutamic acid (LMW-γ-PGA). Traditional methods for producing LMW-γ-PGA, such as enzymatic hydrolysis, often suffer from low operational stability. To address this, a stable and efficient biosynthesis method was developed by overexpressing γ-PGA hydrolase in B. amyloliquefaciens NB. Plasmid pNX01 was constructed using endogenous plasmid p2Sip, exhibiting a low % loss rate of 4% after 100 consecutive passages. This plasmid was used to screen for a highly active PgdS hydrolase, significantly increasing the γ-PGA titer and reducing its molecular weight. As a result, a yield of 17.62 ± 0.38 g/L LMW-γ-PGA, with a weight-average molecular weight of 20–30 kDa, was obtained from direct fermentation of JAT extract. This study offers a promising approach for the commercial production of LMW-γ-PGA. However, further optimization strategies are required to refine the fermentation process and boost production efficiency [138].

5. Other Products from Inulin

Further essential industrial products from inulin-containing materials as feedstock are inulinases themselves [29,30,139,140], fructose and high-fructose syrup [15,141], prebiotic fructooligosaccharides (FOS), single-cell protein, and single-cell oil [142]. The most successful biotechnological processes for synthesizing inulinases and optimizing enzyme kinetic parameters have been developed recently for A. terreus, K. marxianus, and Rhizopus oryzae [31,141,142,143]. Fructose and fructose syrups are used in the food industry and medicine because fructose has a low glycemic index and is desirable for improving the sweetening of candies, chocolates, ice cream, etc. Prebiotic FOS (non-cariogenic, indigestible compounds) can replace sucrose in food formulations due to their lower sweetness property [144,145,146]. Fructose is obtained from inulin after exoinulinase treatment and during LA fermentation of inulin [3]. Endo-inulinase treatment of inulin-containing feedstocks profitably produces HFS and FOS [139].

Another valuable metabolite that is obtained from JAT extract is gluconic acid. The process engages co-immobilized Zymomonas mobilis and inulinase from A. niger by continuous cultivation with a yield of 23.4 g/L/h [147]. Actinobacillus succinogenes converting JAT extract yielded 52.7 g/L of succinic acid during batch cultivation [148], while Propionibacterium acidipropionici accumulated 26.2 g/L of propionic acid by a fed-batch process using the same substrate [149]. As much as 117.5 g/L poly-malic acid was obtained by Aureobasidium pullulans from JAT extract and during the fed-batch process [150]. Therefore, future perspectives in producing multiple valuable biochemicals from inulin may focus on optimizing fermentation conditions, increasing the efficiency of bacterial strains, and developing sustainable, cost-effective methods.

Figure 3 presents an overview of the biochemical synthesis pathways for the valuable products discussed above.

6. Conclusions and Perspectives

This overview of the bioconversion processes of inulin to valuable products shows that the methods for the synthesis of biofuels have the closest industrial application. Both natural and genetically improved strains of yeast (producing ethanol) and bacteria (producing 2,3-butanediol) achieved high productivity of the target metabolite and yields very close to the theoretical maximum. Furthermore, the concentrations of the products obtained (about 100 g/L for ethanol and over 120 g/L for 2,3-BD) are sufficient for profitable subsequent extraction steps. On the contrary, biobutanol was obtained from inulin in low concentrations (below 13–14 g/L). Of the acids, the success of lactic acid production was tremendous, with a concentration of over 140 g/L, a degree of substrate conversion between 94 and 98%, and a productivity of over 4.3 g/L/h. Citrate biosynthetic processes are also promising for relevant biotechnology development, with a yield of 200 g/L, productivity of 0.51 g/L/h, and yield of 0.85 g/g inulin-containing substrate. When evaluating raw inulin materials, the most promising inulin-rich substrate is JAT, followed by chicory. Moreover, the areas and yields of artichokes are significantly greater than those of chicory because the latter has a lower market price.

Integrating inulin-based processes into existing biorefineries could diversify output and maximize resource utilization. Hybrid biorefineries, capable of processing multiple feedstocks, would improve overall economic viability by leveraging infrastructure for simultaneous or sequential processing. Commercialization efforts would benefit from pilot-scale and demonstration projects, proving large-scale inulin-based biorefineries’ feasibility and economic potential. In conclusion, inulin is competitive compared to other renewable substrates, although still more expensive compared to molasses and lignocellulosic residues. Addressing economic challenges, such as inulin’s market availability, competitive pricing, and production scalability, is critical for transitioning inulin-based biorefineries from experimental to industrial scales. Future biorefinery models can capitalize on inulin’s potential by using waste streams, such as agricultural residues rich in inulin, thereby reducing raw material costs and promoting circular economy principles. Utilizing agricultural byproducts can provide a steady, low-cost inulin source while creating additional revenue streams for farmers and reducing waste. Leveraging inulin as a biorefinery substrate could facilitate a sustainable transition toward bio-based products, providing advancements in microbial and enzyme technology, process integration, and economic feasibility. Further research into optimizing inulin hydrolysis and developing specialized biorefineries could position inulin as a central player in green chemistry and industrial biotechnology.