The study, published in MicrobiologyOpen, was led by scientists at Teagasc, the Irish agriculture and food development authority, working with strains from their culture collection and the German Collection of Microorganisms and Cell Cultures. Their motivation was twofold. First, the global mannitol market, valued at roughly 451 million US dollars in 2024 and growing at about 5 percent annually, depends on a process that consumes energy and generates an unwanted co-product. Second, microbial fermentation offers an inherently gentler alternative: it runs at atmospheric pressure, at mild temperatures between 30 and 37 degrees Celsius, and can in principle use cheap agricultural feedstocks instead of pure sugars. The catch has always been that complex plant carbohydrates usually need to be broken down into fructose before microbes can turn them into mannitol, adding a costly processing step.

The Irish team’s insight was to pick a feedstock whose fructose chains are short enough for the bacteria to handle on their own. Yacon, Smallanthus sonchifolius, is a tuber native to the Andes that stores much of its carbohydrate as fructooligosaccharides, or FOS, polymers of fructose with a degree of polymerization typically no greater than ten. The researchers obtained yacon root powder, prepared by spray-drying yacon syrup from a grower in County Kilkenny, Ireland, where the crop has been trialed successfully in temperate conditions. Chemical analysis showed the powder contained 54.8 percent FOS by weight, along with 31.1 percent free sugars, mostly fructose, glucose and sucrose. Chromatographic profiling revealed that most of the FOS chains carried between four and six fructose units, making them considerably less complex than the inulin found in Jerusalem artichoke or chicory.

To ferment this material, the team screened ten strains of fructophilic lactic acid bacteria, or FLAB, belonging mainly to the genera Leuconostoc and Fructobacillus, all previously isolated from flowers on an Irish farm, including cotoneaster, dandelion and white clover blossoms. These bacteria thrive in fructose-rich environments such as nectar and are known to tolerate fructose concentrations of up to 40 percent. Crucially, genome analysis of one strain, Leuconostoc mesenteroides DPC7261, identified two genes encoding glycoside hydrolases carrying the GH32 domain, the enzymatic signature of invertases and inulinases, suggesting the bacteria could cleave yacon FOS into fructose themselves. That endogenous enzymatic toolkit is what makes a single-step, consolidated bioprocess possible.

The screening results were striking. When grown for 20 hours in laboratory medium containing 1 percent yacon root powder at 30 degrees Celsius, all ten flower-derived FLAB strains produced between 4 and 5 grams of mannitol per liter. The official type strains of the same species fared noticeably worse, producing less than 3 grams per liter, and the gap was especially pronounced for Fructobacillus fructosus, where the flower isolates yielded roughly twice as much mannitol as the type strain. A comparison strain, Limisolactobacillus fermentum, which has been reported to make mannitol from pure fructose, managed less than 3 grams per liter on yacon powder, even though it produced over 10 grams per liter when given plain fructose. The flower isolates, in other words, were uniquely suited to the untreated FOS substrate.

From the screen, the researchers focused on two strains: Fructobacillus fructosus DPC 7237, from white clover flowers, and Leuconostoc mesenteroides DPC 7246, from cotoneaster flowers. Although the Fructobacillus strains produced slightly higher peak titers, they flocculated strongly regardless of agitation, complicating cell density measurements and consistent inoculation. Leuconostoc mesenteroides DPC 7246 grew more robustly and was less demanding about oxygen levels, so it became the workhorse for optimization and scale-up. Time-course experiments showed mannitol production peaked between 16 and 20 hours, reaching 5.1 grams per liter for DPC 7246 and 5.5 grams per liter for DPC 7237, before declining after 24 hours. The researchers suggest that because the mannitol dehydrogenase reaction is reversible, the bacteria may reconsume mannitol to support central metabolism or regenerate cofactors once fructose runs low.

The team then tested whether temperature, agitation or substrate loading could push titers higher. Raising the temperature to 37 degrees Celsius or removing agitation brought no significant improvement, so subsequent work proceeded at 30 degrees Celsius with mild shaking. Doubling the yacon powder concentration to 2 percent increased peak mannitol by only about a third, and the extra substrate shifted the bacterium’s metabolism: ethanol appeared as a by-product, lactic acid rose, acetic acid fell, and the yield of mannitol per gram of FOS dropped from 0.92 to 0.635 grams. This overflow metabolism, in which surplus fructose is routed through the pentose phosphate pathway and ultimately into ethanol, suggests that simply loading more substrate is not enough; future processes will likely need fed-batch strategies to keep fructose flowing at a rate the cells can channel into mannitol.

The critical test came at scale. Moving from 15-milliliter tubes to 200-milliliter benchtop bioreactors, with controlled heating and mixing but no aeration and no pH adjustment, the researchers found that mannitol production peaked at 6.7 grams per liter, essentially unchanged from small-scale results, and actually arrived earlier, at 24 to 28 hours rather than 32. A post-hoc comparison across volumes showed titers of 6.55 grams per liter at 15 milliliters and 6.38 grams per liter at 200 milliliters at the 24-hour mark, both exceeding the 5.73 grams per liter seen in 40-milliliter shake flasks, where extra aeration appears to have altered the cells’ redox balance and reduced their need to regenerate NAD+ via mannitol. Accounting for the FOS content of the powder, the peak yields at bioreactor scale were 0.61 to 0.62 grams of mannitol per gram of FOS at both temperatures tested.

Those yields stand up well against the published literature on complex substrates. Fermentation of hydrolyzed inulin has delivered 0.58 grams per gram, chicory-derived inulin hydrolysate 0.69 grams per gram, and Jerusalem artichoke extract 0.68 grams per gram, but all of those required the feedstock to be saccharified before fermentation. Uncracked, unhydrolyzed substrates have generally performed far worse: Jerusalem artichoke juice fermented directly by Lactobacillus casei strains yielded only about 3.6 grams of mannitol per liter, compared with the 4 to 5 grams per liter the Irish strains achieved from yacon powder at screening concentrations. The researchers attribute this advantage to the short chain length of yacon FOS, which allows the bacteria’s own enzymes to hydrolyze and ferment the substrate simultaneously, a genuine consolidated bioprocess.

The work remains a proof of concept, and the authors are candid about the hurdles ahead. Titer and productivity must rise substantially to compete industrially, which will demand higher substrate concentrations, strains with greater fructose tolerance, cheaper nitrogen sources such as tryptone or corn steep liquor to replace the complex laboratory medium, and feeding strategies that suppress ethanol formation. Yet the underlying proposition is compelling: a natural, plant-based fructooligosaccharide from a tuber that grows well in temperate climates, converted by food-safe bacteria at mild temperatures into a sweetener with a low glycaemic index and applications spanning food and pharmaceuticals, all in one vessel and one step. As demand grows for sugar substitutes that do not carry the metabolic baggage of sucrose, the humble yacon tuber and its flower-borne microbial partners may have earned a place in the industrial biotechnology spotlight.

Subject of Research: Microbial production of mannitol from yacon fructooligosaccharides using fructophilic lactic acid bacteria

Article Title: Single‐Step Production of Mannitol From Yacon Fructo‐Oligosaccharides Using Fructophilic Lactic Acid Bacteria

Article References: Rajkumar, A. S., Leech, J., & McAuliffe, O. (2026). Single‐Step Production of Mannitol From Yacon Fructo‐Oligosaccharides Using Fructophilic Lactic Acid Bacteria. MicrobiologyOpen, 15(5), Article e70387. https://doi.org/10.1002/mbo3.70387

Image Credits: AI Generated

DOI: 10.1002/mbo3.70387

Keywords: mannitol, yacon, fructooligosaccharides, fructophilic lactic acid bacteria, Leuconostoc mesenteroides, Fructobacillus fructosus, fermentation, biotechnology, sweeteners, consolidated bioprocessing, prebiotics, sustainable food production

The new strains, branded Cellusec®, were built on an industrial lignocellulosic bioethanol yeast chassis rather than a laboratory strain, a distinction the authors emphasize as critical for practical application. Industrial ethanol yeasts must tolerate the inhibitors, osmotic stress, and high ethanol concentrations characteristic of real fermentation broths, traits that are often lost when laboratory strains are used as engineering platforms. By starting from a proven industrial background and incrementally adding secretion capacity, the team ensured that the final strains could still deliver the fermentation performance expected in a plant setting.

The genetic architecture of the engineered strains reflects the biochemistry of cellulose itself. Crystalline cellulose cannot be dismantled by any single enzyme; it requires the coordinated action of three complementary activities. Cellobiohydrolases CBHI and CBHII processively peel chains from the crystalline polymer, releasing the disaccharide cellobiose. Endoglucanases (EG) nick the polymer internally, generating new chain ends for the cellobiohydrolases to attack. Finally, β-glucosidase (BGL) cleaves accumulated cellobiose into glucose, simultaneously relieving the product inhibition that otherwise slows the cellobiohydrolases. The researchers inserted a single copy each of CBHI, CBHII, and EG encoding genes together with three copies of a BGL gene, tuning the dosage to reflect the particular sensitivity of the system to β-glucosidase availability.

A notable methodological strength of the study is that every intermediate strain was evaluated along the engineering path. Rather than building the final construct and testing it blindly, the team measured performance after each successive genetic modification, exposing trade-offs that would otherwise have remained hidden. This stepwise audit revealed how added secretion burdens interact with the strain’s native fermentation physiology, informing which combinations of secreted enzymes deliver net benefit and which impose costs that outweigh their contribution to saccharification.

To push secretion capacity further, the researchers overexpressed HAC1i, the active, spliced form of the transcription factor that governs the unfolded protein response in yeast. HAC1i activation expands the endoplasmic reticulum’s folding capacity and upregulates components of the secretory pathway, allowing the engineered cells to export substantially more enzyme protein without succumbing to secretion stress. This layer of engineering, stacked on top of the cellulase gene inserts, proved decisive in achieving supernatant enzyme activities high enough to matter industrially.

The experimental demonstrations were designed to mirror the conditions that actually constrain commercial plants. In saccharification assays using crystalline cellulose, cell-free culture supernatant from the Cellusec® strains reduced the required exogenous cellulase cocktail by up to 53 percent when tested at low temperature and sub-saturating enzyme loading—the very regime in which industrial operations seek to economize. The result demonstrates that secreted enzymes are not merely a laboratory curiosity but a genuine substitute for a meaningful fraction of purchased enzyme, translating directly into operational cost reduction.

Two real-world feedstocks provided the acid test. The first was furfural residue (FR), the lignocellulosic leftover from industrial furfural manufacturing, a material that is abundant and cheap but recalcitrant to enzymatic attack. In simultaneous saccharification and fermentation (SSF) of furfural residues, the cellulase-secreting strains achieved ethanol titers up to 14 percent higher than the non-secreting parent strain under otherwise identical, enzyme-limited conditions. The improvement indicates that the secreted enzymes complement the commercial cocktail at precisely the loadings where plants would like to cut spending.

The second feedstock delivered an even stronger result. Pretreated softwood from forestry waste (FW) is among the most challenging cellulosic substrates, with a composition and inhibitor profile that punish conventional SSF processes. Working with the commercial preparation CTec3-HS, the Cellusec® strains reached ethanol titers of up to 5.15 percent by volume, a 25 percent improvement over the non-secreting parent under the same conditions. For an industrial process, a quarter more ethanol from the same substrate and enzyme budget represents a difference between marginal and competitive economics.

Perhaps the most forward-looking finding concerns enzyme synergy and its substrate dependence. The researchers observed distinct, feedstock-specific synergies between CTec3-HS and the various secreted enzymes, underscoring that there is no universal best enzyme combination. Notably, the team expressed the recently discovered processive endoglucanase RfGH5_4 in S. cerevisiae for the first time. This enzyme displayed particularly strong synergy with CTec3-HS during forestry waste SSF, suggesting that next-generation secreted enzyme cocktails can be rationally tailored to specific feedstocks rather than relying on one-size-fits-all commercial blends. The first-ever yeast expression of RfGH5_4 also expands the enzymatic toolbox available for strain engineers working on consolidated bioprocessing.

The work represents a pragmatic milestone on the road to consolidated bioprocessing (CBP), the long-sought configuration in which a single microorganism both produces the saccharifying enzymes and ferments the released sugars to ethanol. Full CBP remains difficult because no known organism combines high cellulolytic capacity with industrial-grade fermentation performance. The Cellusec® strategy instead charts an intermediate course: a fermentation-competent industrial yeast that contributes part of the enzyme load while still relying on a reduced dose of commercial cocktail. The authors conclude that cellulase-secreting yeast can substantially reduce commercial enzyme requirements in SSF setups, and that further gains will come from tailoring secreted enzyme combinations to particular feedstocks and optimizing the composition of the secreted cocktail itself. As second-generation ethanol plants worldwide contend with enzyme costs that can make or break profitability, the prospect of yeast factories that brew their own catalysts alongside the fuel is a development the bioeconomy will be watching closely.

Subject of Research: Engineering cellulase-secreting Saccharomyces cerevisiae to reduce commercial enzyme requirements in lignocellulosic bioethanol production

Article Title: Cellulase-secreting yeast can substantially reduce commercial enzyme requirements in lignocellulosic ethanol production

Article References: Thevelein, B., Demeke, M. M., Desmet, T., & Thevelein, J. M. (2026). Cellulase-secreting yeast can substantially reduce commercial enzyme requirements in lignocellulosic ethanol production. Biotechnology for Biofuels and Bioproducts. https://doi.org/10.1186/s13068-026-02810-1

Image Credits: AI Generated

DOI: 10.1186/s13068-026-02810-1

Keywords: cellulase secretion, second-generation bioethanol, Saccharomyces cerevisiae, lignocellulosic biomass, simultaneous saccharification and fermentation, consolidated bioprocessing, cellobiohydrolase, endoglucanase, beta-glucosidase, forestry waste, furfural residues, industrial yeast

Lignocellulose is simultaneously one of the most abundant organic materials on Earth and one of the most stubborn to break down. Its two dominant carbohydrate components, cellulose and hemicellulose, differ sharply in architecture. Cellulose consists of long, linear chains of glucose that pack into crystalline microfibrils, while hemicellulose is a branched, chemically heterogeneous tangle of hexose and pentose sugars decorated with side groups such as arabinose and acetyl esters. Any organism hoping to feast on plant biomass must therefore deploy a correspondingly diverse arsenal of carbohydrate active enzymes, or CAZymes, including glycosyl hydrolases that cleave sugar backbones, carbohydrate esterases that strip off side groups, and carbohydrate-binding modules that tether the catalytic machinery to its insoluble target. Understanding which enzymes a bacterium makes, and when it makes them, is central to harnessing it for consolidated bioprocessing, a strategy in which a single organism both degrades biomass and ferments the resulting sugars into fuel.

To probe this capability, the team grew three strains of T. thermosaccharolyticum on a panel of carbohydrates ranging from simple hexoses and pentoses to complex hemicellulosic polysaccharides, then used liquid chromatography–tandem mass spectrometry to quantify the proteins each condition induced. The growth assays revealed a broad substrate palette, confirming that the species can metabolize a wide variety of sugars and oligosaccharides. But the proteomic data told a subtler story: the strains differed substantially in both the abundance of CAZymes they produced and their actual ability to grow on particular polysaccharides. This strain-to-strain variation matters for anyone hoping to select or engineer an industrial chassis, because the ability to digest a substrate on paper does not guarantee that a given isolate will express the right enzymatic toolkit at the right levels.

One of the clearest signals in the dataset emerged when the researchers compared global proteomic responses during growth on hexoses versus pentoses. Cells feeding on pentose sugars, the five-carbon building blocks liberated from hemicellulose, mounted a far stronger expression of proteins dedicated to handling hemicellulose-derived carbohydrates. The authors interpret this as a molecular reflection of structural reality: hemicellulose is more architecturally complex than cellulose, so liberating and metabolizing its sugars demands more enzymatic and transport machinery. For biorefinery design, this suggests that hemicellulose utilization is a distinct physiological program that can be studied, and potentially optimized, independently of cellulose degradation.

Perhaps the most surprising finding concerned arabinose, a pentose that constitutes only a minor fraction of lignocellulose. Despite its modest abundance in plant biomass, arabinose drove elevated CAZyme production at levels comparable to those triggered by complex hemicellulose-derived carbohydrates. In other words, this seemingly minor sugar acts as a powerful regulatory cue, essentially telling the bacterium that hemicellulose is nearby and prompting it to ramp up its degradative apparatus. Such a counterintuitive response hints that T. thermosaccharolyticum uses arabinose as an environmental signal, a strategy that could be exploited to induce enzyme production in industrial fermentations without relying on expensive complex substrates.

The centerpiece of the study, however, is the identification of two polysaccharide utilization loci, or PULs, in this Gram-positive thermophile. PULs were first characterized in gut Bacteroidetes, where they appear as clustered gene sets that coordinate the sensing, import and stepwise degradation of specific polysaccharides. Finding analogous systems in a thermophilic Gram-positive anaerobe is notable; the authors report these as among the first such loci described in thermophilic anaerobes, and they term them gpPULs, for Gram-positive polysaccharide utilization loci. Each gpPUL bundles together the genes encoding CAZymes, sugar transporters and regulatory proteins needed to attack a particular class of hemicellulosic substrate, providing a self-contained module for polysaccharide harvesting.

By measuring proteomic responses across multiple carbohydrates and multiple strains simultaneously, the team could correlate specific enzyme and transporter expression patterns with growth outcomes, allowing them to delineate the boundaries and contents of the two gpPULs and to assign functions to genes whose roles had previously been uncertain. The loci include glycosyl hydrolases targeting xylan backbones, enzymes that process arabinose side chains, carbohydrate-binding modules that recognize the insoluble substrate, and transport systems that funnel the liberated oligosaccharides into the cell. Building on this map, the researchers constructed a model describing how T. thermosaccharolyticum deploys these components to degrade hemicellulose at elevated temperature, from initial substrate recognition through extracellular cleavage to intracellular sugar metabolism.

The thermophilic nature of the organism adds practical weight to these findings. Running industrial bioprocessing at high temperatures offers real advantages: reduced risk of contamination by mesophilic microbes, improved solubility and kinetics of substrates, and easier recovery of volatile products. A thermophile with a well-characterized, genetically defined system for hemicellulose degradation is therefore an attractive platform for consolidated bioprocessing of lignocellulosic feedstocks. Moreover, because the gpPULs are organized as discrete gene clusters, the authors suggest that the identified genes could be transferred into other species to extend their substrate ranges, a form of synthetic biology that could broaden the menu of biomass components that engineered microbes can convert into fuels and chemicals.

The work also speaks to questions beyond the bioreactor. Saccharolytic bacteria play significant roles in human health and in the cycling of carbon through the environment, and the regulatory logic uncovered here, in which minor sugars act as major inducers of degradative machinery, may illuminate how microbial communities partition plant material in soils and guts alike. As the authors note, the study advances understanding of saccharolytic species both in applied settings such as biofuel production and in the biosphere at large. With the genetic blueprints of two thermophilic gpPULs now in hand, researchers have a concrete starting point for engineering faster, more complete conversion of plant waste into renewable energy, turning one of nature’s most recalcitrant materials into a feedstock for the bioeconomy.

Subject of Research: Proteomic characterization of lignocellulosic carbohydrate utilization and polysaccharide utilization loci in the thermophilic bacterium Thermoanaerobacterium thermosaccharolyticum

Article Title: Proteomics and characterization of lignocellulosic carbohydrate utilization for Thermoanaerobacterium thermosaccharolyticum reveals two thermophilic Gram-positive polysaccharide utilization loci

Article References: Stephens, K., Davin, M. E., Giannone, R. J., Bomble, Y. J., Lynd, L. R., Holwerda, E. K., & Hettich, R. L. (2026). Proteomics and characterization of lignocellulosic carbohydrate utilization for Thermoanaerobacterium thermosaccharolyticum reveals two thermophilic Gram-positive polysaccharide utilization loci. Biotechnology for Biofuels and Bioproducts. https://doi.org/10.1186/s13068-026-02822-x

Image Credits: AI Generated

DOI: 10.1186/s13068-026-02822-x

Keywords: Thermoanaerobacterium thermosaccharolyticum, polysaccharide utilization loci, CAZymes, lignocellulose, hemicellulose, biofuels, proteomics, thermophilic anaerobe, consolidated bioprocessing, arabinose, sugar transporters, anaerobic fermentation