Fermentation performance and intracellular metabolite profiling of Fusarium oxysporum cultivated on a glucose–xylose mixture

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Abstract

The influence of aeration on the fermentation of a mixture of glucose and xylose by the naturally xylose fermenting fungus Fusarium oxysporum was studied in batch cultivations. The aeration level had considerable influence on the co-metabolism of glucose and xylose. Under anaerobic conditions xylose consumption was limited and xylitol was the main product of xylose metabolism. When the artificial electron acceptor acetoin was added xylose consumption increased by 95% with a significant improvement of ethanol yield from 0.96 to 1.52 mol/mol, mainly due to a 72% reduction of xylitol excretion. The profile of the phosphorylated intermediates during the fermentation suggests that a high glycolytic flux occurred during the oxygen-limited conditions, characterized by high efflux of glyceraldehyde-3-P (G3P) and fructose-6-P (F6P) from Pentose Phosphate Pathway (PPP) to Embden-Meyerhof-Parnas Pathway (EMP) resulting in the higher ethanol production. An accumulation of sedoheptulose-7-P (S7P) was also observed indicating a block in the PPP which affects the production of NADPH and results in the production of acetate and the reduced xylose consumption.

Introduction

Cellulose and hemicellulose are potential carbon sources for the production of ethanol by fermentation. The composition of the hemicellulose will depend on the raw material, but will in many cases be xylan. Fermentable sugars from the hemicellulose stream will therefore essentially be glucose and xylose and they can be released from lignocellulosics by single- or two-stage hydrolysis, thereby leading to mixtures of glucose and xylose or separate glucose- and xylose-rich streams. Conventional methods, applied for bioconversion of cellulose and hemicellulose to ethanol, involve acid or enzyme hydrolysis of the biopolymers to soluble oligosaccharides followed by fermentation to ethanol. An alternative approach has been a direct process in which one or more microorganisms carry out simultaneous hydrolysis and fermentation in the same bioreactor. A few microbial species such as Neurospora crassa and Fusarium oxysporum have been reported to acquire the ability of fermenting cellulose directly to ethanol in the 1980s. We have earlier reported on the direct conversion of biomass to ethanol by F. oxysporum [1], [2]. Cellulases and xylanases from this microorganism have been characterized [3], [4], [5], [6], [7]. If F. oxysporum is used as the fermentation organism, it is not necessary to perform a separate enzymic hydrolysis of the lignocellulosic raw material, as this microorganism can produce the necessary enzymes.

Xylitol is a major by-product formed during anaerobic and oxygen-limited fermentation of xylose by many natural xylose-fermenting eukaryotic microorganisms [8], [9], [10]. This lowers the ethanol yield in ethanolic fermentation of lignocellulosic hydrolysates and reduces the economic feasibility of a future process [11]. Xylitol formation has been ascribed to the difference in cofactor preference for the two initial enzymes in the conversion of ethanol [12]. The first enzyme, xylose reductase, EC 1.1.1.21 (XR), which reduces xylose to xylitol [13] uses NADPH or NADH as cofactor, whereas the second enzyme, xylitol dehydrogenase, EC 1.1.1.14 (XDH), almost exclusively uses NAD+ in the oxidation of xylitol into xylulose so that xylitol excretion occurs as a result of NAD+ depletion [14], [15], [16].

Micro-organisms adapt to changes in their environment by adjusting their metabolic activities; enzyme levels and activities are altered, leading to changes in intracellular metabolite concentrations. Metabolite concentrations are very sensitive to small variations in growth conditions, and, usually, turnover rates of metabolites are high and concentrations are low. To ascertain that the experimental data reflect the actual intracellular concentrations as well as possible, sampling of cells and stopping of metabolism should be rapid and instantaneous, and extraction of the investigated metabolites should be complete. These prerequisite have been shown to all be fulfilled in an extraction method for yeast cells, reported by Koning and van Dam [17], and it has already found application in a number of studies. The method allows for rapid quenching of metabolism by spraying cells into −40 °C methanol/water (60%, v/v) and extraction of metabolites at neutral pH.

In the present work, the fermentation capabilities of F. oxysporum, were evaluated in minimal medium under aerobic, oxygen-limited and anaerobic conditions in terms of growth, substrate consumption, product and by-product formation during batch growth. The metabolite levels for the phosphorylated intermediates from Pentose Phosphate (PP), Embden-Meyerhof-Parnas (EMP), and the glycolytic pathway were determined.

Thus, besides providing an insight into the little investigated co-metabolism of glucose and xylose in F. oxysporum, these investigations might point towards the metabolic conditions required for efficient xylose consumption.

Section snippets

Microorganism and culture conditions

F. oxysporum F3, isolated from cumin [1] was used. The stock culture was maintained on potato–dextrose–agar at 4 °C. For the growth phase under aerobic conditions (1 vvm), a mineral medium was used as described previously [4]. A 5 mL mycelia and spore suspension of F. oxysporum from a 6 day old culture, grown on a PDA slope at 30 °C, was inoculated to five 500 mL Erlenmeyer flasks each containing 200 mL of the above mentioned mineral medium. A glucose–xylose mixture (10 g/L of each sugar) was used as

Aerobic growth phase of F. oxysporum on glucose/xylose substrate mixture

Aerobic growth of F. oxysporum was carried out with an aeration of 1 vvm. The concentrations of glucose, xylose and the (dry) cell mass during the time course of the cultivation are shown in Fig. 1. Glucose was consumed preferentially to xylose. Glucose exhaustion occurred at 24 h. The specific glucose consumption rate was 0.94 mmol/g DW/h. Afterwards, the consumption of d-xylose began and the specific xylose consumption rate found to be 0.53 mmol/g DW/h. The yield of biomass on sugars was 0.32 (g

Conclusions

In conclusion, in this study we examined the conversion of a mixture of glucose and xylose to ethanol by the natural xylose-fermenting fungus F. oxysporum, under different aeration levels and in the presence of acetoin. The addition of acetoin led to a 95% higher xylose consumption after 120 h of anaerobic fermentation, while concomitant significant increase of ethanol yield from 0.96 to 1.52 mol/mol was found, mainly due to a 72% reduction of xylitol excretion. We also examined the differences

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