Fermentation is the process by which cells break down sugar without oxygen, producing alcohol and carbon dioxide. In the 19th century people thought only living cells could do this mysterious job. Harden crushed yeast cells and proved that the filtered “yeast juice” still fermented. He also found that adding inorganic phosphate sped the reaction up, showing phosphate’s key role in enzyme activity. Von Euler-Chelpin isolated a helper molecule—later called NAD⁺—that re-activated the reaction and coined the idea of a coenzyme. Their work established that fermentation is a chain of chemical steps, a metabolic pathway. The classic glycolysis diagram in today’s textbooks begins with results from that era. Food industries now fine-tune temperature and nutrients to optimize fermentation and mass-produce bread, beer, and miso. In medicine, missing enzymes or coenzymes hint at metabolic diseases. Thus their achievements have huge value for both everyday life and large-scale industry.

In 1906 Harden and Young reported that adding inorganic phosphate to cell-free yeast extract (zymase) greatly accelerated sugar breakdown and produced soluble phosphate esters. The product was later identified as fructose-1,6-bisphosphate, hinting at the first energy-investment step in glycolysis. By measuring CO₂ and glycerol they confirmed the stoichiometry of two moles CO₂ and two moles ethanol per mole of glucose. Von Euler-Chelpin separated a heat-labile low-molecular fraction called “cozymase” that restored fermentation, and in the 1920s he deduced it was a pyridine dinucleotide, later named nicotinamide adenine dinucleotide (NAD⁺). This showed that electron-carrying coenzymes drive metabolic sequences and laid foundations for bioenergetics. Their achievements linked to the work of Warburg, Embden, and Meyerhof, helping complete what is now known as the Embden–Meyerhof–Parnas pathway. The step-wise energy-conversion model of fermentation also guided discovery of ATP and phosphorylated intermediates, deepening our grasp of cellular energy capture. Today the insights extend to yeast metabolic engineering, biofuel production, and quality control of lactic-acid foods. Glycolytic enzyme inhibitors are also explored as drugs that target cancer metabolic reprogramming. The 1929 Nobel Prize in Chemistry honored both the fundamental and practical value of these discoveries.

Harden quantified in-vitro fermentation of cell-free yeast extract using micro gas burettes, time-resolving CO₂ evolution under anaerobic low-pO₂ conditions. He observed a phosphate-dependent acceleration of the initial rate and purified the concomitantly formed acid-soluble phosphate ester on anion-exchange columns, later reporting with Haberland that the compound was hexose-2,6-bisphosphate. Von Euler-Chelpin dialyzed heat-inactivated zymase, isolated the low-molecular fraction by nitrosation chromatography, and identified cozymase as the factor restoring coenzyme activity. Subsequent work showed cozymase possessed a pyridine-nucleotide backbone, paving the way for Karrer’s crystallization of NAD⁺ in 1929. Harden’s stoichiometric measurements indicated a 2:1 ratio of CO₂ evolution to phosphate consumption, providing the basis for the phosphate-cycle model in yeast fermentation. Their data supplied crucial experimental support for formulating the AB steps of the Embden–Meyerhof–Parnas pathway. Von Euler-Chelpin’s inhibition studies with dinitrofluorene derivatives suggested the active locus of the coenzyme resided at the N-position of the purine/pyridine ring. These insights underpinned the Lohmann–Meyerhof concept of metabolic energy transfer via redox-potential differentials. To this day, cell-free reconstituted fermentation is being re-examined as an ATP-free production platform in synthetic biology. Harden and von Euler-Chelpin’s methodologies provided the stoichiometric perspective that presaged metabolomics and flux-balance analysis, directly influencing mathematical models in metabolic engineering. Their work defined the separation between enzymology and biochemistry, and proved that energy conversion can occur in membrane-free systems, a principle still cited in modern biocatalysis theory.