Fermentation, cell-free

E. Buchner (Berlin) biochemical researches and the discovery of cell-free fermentation. 

Cell-free fermentation broth contains 8 X 10 5 mol/1 immunoglobulin C. Ninety per cent of antibody is recovered by adsorption on non-polar resin. 

Experiments in 500 ml Erlenmeyer flasks and Fernbach flasks contained 200 ml and 1 L of EPl and EP2 medium respectively. Inocuia added to these cultures was 2 ml of spore suspension (5.0 optical density at 540 nm) for each 100 ml EP medium. All cultures were grown at 37°C in a shaking incubator (New Brunswik Sci. Co., USA), at 200 rpm. Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic assays and extracellular protein determinations by the Lowry method (14). Experiments in the 14 L fermentor (Microgen Fermentor New Brunswik Sci. Co., USA) were carried with lOL of fermentation medium EP2 and inoculum added was IL of mycelium grown 24 h in... 

Determinations were carried out in cell free filtrates obtained at 96 h of fermentation on 1% lemon peel. 

Hexose diphosphate was found by Harden and Young69 in cell-free alcoholic-fermentation liquors. In 1930, it was observed that addition of fluoride to fermenting-yeast extracts leads to an accumulation of 0-phospho-D-glyceronic acid,60 which is also a metabolite of muscle extracts.61 Attention was turned, therefore, to the pathway from hexose diphosphate to 0-phos-pho-D-glyceronic acid. In 1932, Fischer and Baer62 described the synthesis of D-glycerose 3-phosphate, and, in 1933, Smythe and Gerischer63 noted... 

All of the principles and ideas covered in the previous section may be translated directly to the use of microorganisms as tools in the production of compounds of plant biosynthetic or biodegradative importance. Just as one finds microbial systems to be of value in preparing metabolites in mammalian systems, it may be possible to use microbial transformations to prepare derivatives of alkaloids that might be found rarely or only in very small quantities in plants. In this way, abundant prototype alkaloids may be used as microbial transformation substrates to provide a range of metabolites. As in the mammalian case, metabolism studies using plant tissues, tissue cultures, or cell-free extracts may be conducted in parallel with microbial metabolic systems. Metabolites common to both would be prepared in quantity by relatively simple fermentation scale-up methods. 

Fermentation is a process in which chemical changes are brought about in an organic substrate through the action of enzymes whether or not these enzymes occur within the cell or are cell free. Air is needed for metabolism, so the process is aerobic.. 

Wohler s preparation of urea from ammonium cyanate, which could in principle be derived totally from inorganic constituents, is cited as an early demonstration (1828) that living cells were not obligatorily required for the synthesis of natural products. I can prepare urea without requiring a kidney or an animal—either man or dog. Three years after the death of Pasteur the finding by Hans and Edouard Buchner (1897) that fermentation still occured in a cell-free extract from yeast and so did not require the presence of organized cells, was virtually the final nail in the coffin for vitalism and an essential preliminary to the study of intermediary metabolism (Chapter 4). 

The most confusing aspect of the pathway proposed by Ochoa and his group now rests with the NAD requirement. In proceeding from L-malic acid to L-lactic acid, there is no net change in oxidation state. Yet in whole cells or cell-free extracts, the malo-lactic fermentation will not proceed in the absence of NAD. Therefore, by the proposed mechanism, one is unable to demonstrate the appearance of reduced cofactor, and the NAD specificity cannot be explained as a redox requirement. However, in the time since this mechanism was proposed, an NAD dependent enzyme (glyceraldehyde-3-phosphate dehydrogenase) has been described which requires NAD in a non-redox capacity (29), and it is possible that the same is true for the enzyme causing the malic acid-lactic acid transformation. 

Edward Buchner Chemistry Biochemistry, cell-free fermentation... 

In 1878 the term enzyme, Greek for "in yeast," was proposed (8). It was reasoned that chemical compounds capable of catalysis, ie, ptyalin (amylase from saliva), pepsin, and others, should not be called ferments, as this term was already in use for yeast cells and other organisms. However, proof was not given for the actual existence of enzymes. Finally, in 1897, it was demonstrated that cell-free yeast extract ("zymase") could convert glucose into ethanol and carbon dioxide in exacdy the same way as viable yeast cells. It took some time before these experiments and deductions were completely understood and accepted by the scientific community. 

Gibberellic acid production was studied in different fermentation systems. Free and immobilized cells of Gibberella fujikuroi cultures in shake-flask, stirred and fixed-bed reactors were evaluated for the production of gibberellic acid (GA3). Gibberellic add production with free cells cultured in a stirred reactor reached 0.206 g/L and a yield of 0.078 g of GA3/g biomass. 

Figure 5 shows some data referring to the ability of biosurfactant to emulsify kerosene produced by B. subtilis ATCC 6633 at the different substrate concentrations tested (5, 10, 20, and 40 g/L). Besides a decrease in surface tension, stabilization of hydrocarbon/water is frequently used as an indicator of surface activity. Note, however, that the quantity of biosurfactant produced should not be related to the E24 because that is an intrinsic property of the molecule. A similar behavior of the emulsifying activity in relation to the carbon source concentration and to the incubation period has been observed. The diverse initial concentrations of commercial sugar studied favor the formation of a surface-active compound, with an emulsifying activity >50% in a 48-h process. The maximum values for emulsion activity of 57.9 and 56.9% were determined for 10 and 20 g/L of substrate, respectively. It should be emphasized that there was a reduction in the E24 after a 96-h period of incubation. Carvalho et al. (36) reported similar results for cell-free fermented broth by Bacillus sp. emulsified in kerosene. 

In true fermentation, the free energy drop between substrate (say glucose) and anaerobic end products is always modest by comparison with respiration, because fermentation is never based on electron transfer chains coupled to phosphorylation. Rather, true fermentations depend upon a variety of oxidation-reduction reactions involving organic compounds, C02, molecular hydrogen, or sulfur compounds. All these reactions are inefficient in terms of energy yield (moles ATP per mole substrate fermented), and, therefore, the mass of cells obtainable per mole of substrate is much smaller than with respiratory-dependent species. 

A bioreactor is a vessel in which biochemical transformation of reactants occurs by the action of biological agents such as organisms or in vitro cellular components such as enzymes. This type of reactor is widely used in food and fermentation industries, in waste treatment, and in many biomedical facilities. There are two broad categories of bioreactors fermentation and enzyme (cell-free) reactors. Depending on the process requirements (aerobic, anaerobic, solid state, immobilized), numerous subdivisions of this classification are possible (Moo-Young, 1986). 

Heterofermentative LAB have the capability to utilize high concentrations of fructose such that the mannitol concentration in the fermentation broth could reach more than 180g/L, which is enough to be separated from the cell-free fermentation broth by cooling crystallization. Lactic and acetic acids can be recovered by electrodialysis (Soetaert et al., 1995). The enzyme mannitol dehydrogenase responsible for catalyzing the conversion of fructose to mannitol requires NADPH (NADH) as cofactor. Thus, it is possible to develop a one-pot enzymatic process for production of mannitol from fructose if a cost-effective cofactor regeneration system can be developed (Saha, 2004). The heterofermentative LAB cells can be immobilized in a suitable support, and... 

E. Buchner. Cell-free fermentation. Nobel Lecture, December 11, 1907. 

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