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Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli.

Bioethanol production from lignocellulosic raw-materials requires the hydrolysis of carbohydrate polymers into a fermentable syrup. During the hydrolysis of hemicellulose with dilute acid, a variety of toxic compounds are produced such as soluble aromatic aldehydes from lignin and furfural from pentose destruction. In this study, we have investigated the toxicity of representative aldehydes (furfural, 5-hydroxymethlyfurfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) as inhibitors of growth and ethanol production by ethanologenic derivatives of Escherichia coli B (strains KO11 and LY01). Aromatic aldehydes were at least twice as toxic as furfural or 5-hydroxymethylfurfural on a weight basis. The toxicities of all aldehydes (and ethanol) except furfural were additive when tested in binary combinations. In all cases, combinations with furfural were unexpectedly toxic. Although the potency of these aldehydes was directly related to hydrophobicity indicating a hydrophobic site of action, none caused sufficient membrane damage to allow the leakage of intracellular magnesium even when present at sixfold the concentrations required for growth inhibition. Of the aldehydes tested, only furfural strongly inhibited ethanol production in vitro. A comparison with published results for other microorganisms indicates that LY01 is equivalent or more resistant than other biocatalysts to the aldehydes examined in this study.

Aldehydes↗

On-line control of fed-batch fermentation of dilute-acid hydrolyzates.

Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).

Biomass↗

Effects of Ca(OH)(2) treatments ("overliming") on the composition and toxicity of bagasse hemicellulose hydrolysates.

Hemicellulose syrups from dilute sulfuric acid hydrolysates of hemicellulose contain inhibitors that prevent efficient fermentation by yeast or bacteria. It is well known that the toxicity of these hydrolysate syrups can be ameliorated by optimized "overliming" with Ca(OH)(2). We have investigated the optimization of overliming treatments for sugar cane bagasse hydrolysates (primarily pentose sugars) using recombinant Escherichia coli LY01 as the biocatalyst. A comparison of composition before and after optimal overliming revealed a substantial reduction in furfural, hydroxymethylfurfural, and three unidentified high-performance liquid chromatography (HPLC) peaks. Organic acids (acetic, formic, levulinic) were not affected. Similar changes have been reported after overliming of spruce hemicellulose hydrolysates (Larsson et al., 1999). Our studies further demonstrated that the extent of furan reduction correlated with increasing fermentability. However, furan reduction was not the sole cause for reduced toxicity. After optimal overliming, bagasse hydrolysate was rapidly and efficiently fermented (>90% yield) by LY01. During these studies, titration, and conductivity were found to be in excellent agreement as methods to estimate sulfuric acid content. Titration was also found to provide an estimate of total organic acids in hydrolysate, which agreed well with the sum of acetic, levulinic, and formic acids obtained by HPLC. Titration of acids, measurement of pH before and after treatment, and furan analyses are proposed as relatively simple methods to monitor the reproducibility of hydrolysate preparations and the effectiveness of overliming treatments.

Biotechnology↗

Effects of some hydrocolloids and water activity on nonenzymic browning of concentrated orange juice.

5-hydroxymethyl furfural (HMF) accumulation and brown pigment formation (BPF) were studied in the presence and absence of some hydrocolloids in concentrated orange juice. The influence of addition of 1.5% xanthan gum (XG) and 1.5% XG and 0.5% microcrystalline cellulose (MCC) were observed on the nonenzymic browning reactions in concentrated orange juice at 45, 60, and 75 degrees C. The effect of water activity was also observed by replacing the samples into different water activity media (0.11 +/- 0.01, 0.30 +/- 0.01, 0.50 +/- 0.01, 0.75 +/- 0.01, and 0.80 +/- 0.01). It was observed that the samples containing 1.5% XG and 0.5% MCC showed the lowest BPF and HMF accumulation, whereas the browning rate and HMF accumulation in pure orange juice were the highest at all temperatures. Maximum BPF and HMF accumulation were observed in the range of water activity between 0.30 and 0.75. The lowest values were observed at aw 0.80.

Beverages↗

Some aromatic hydrazone derivatives as inhibitors for the corrosion of C-steel in phosphoric acid solution.

The effect of furfural benzoylhydrazone and its derivatives (I-VII) as corrosion inhibitors for C-steel in 1M phosphoric acid solution has been studied by weight-loss and galvanostatic polarization techniques. A significant decrease in the corrosion rate of C-steel was observed in the presence of the investigated inhibitors. This study revealed that, the inhibition efficiency increases with increasing the inhibitor concentration, and the addition of iodide ions enhances it to a considerable extent. The effect of temperature on the inhibition efficiency of these compounds was studied using weight-loss method. Activation energy (E(a)*) and other thermodynamic parameters for the corrosion process were calculated and discussed. The galvanostatic polarization data indicated that, the inhibitors were of mixed-type, but the cathode is more polarized than the anode. The adsorption of these compounds on C-steel surface has been found to obey Frumkin's adsorption isotherm. The mechanism of inhibition was discussed in the light of the chemical structure of the undertaken inhibitors.

Adsorption↗

Effects of furfural on anaerobic continuous cultivation of Saccharomyces cerevisiae.

Furfural is an important inhibitor of yeast metabolism in lignocellulose-derived substrates. The effect of furfural on the physiology of Saccharomyces cerevisiae CBS 8066 was investigated using anaerobic continuous cultivations. Experiments were performed with furfural in the feed medium (up to 8.3 g/L) using three different dilution rates (0.095, 0.190, and 0.315 h(-1)). The measured concentration of furfural was low (< 0.1 g/L) at all steady states obtained. However, it was not possible to achieve a steady state at a specific conversion rate of furfural, q(f), higher than approximately 0.15 g/g.h. An increased furfural concentration in the feed caused a decrease in the steady-state glycerol yield. This agreed well with the decreased need for glycerol production as a way to regenerate NAD+, i.e., to function as a redox sink because furfural was reduced to furfuryl alcohol. Transient experiments were also performed by pulse addition of furfural directly into the fermentor. In contrast to the situation at steady-state conditions, both glycerol and furfuryl alcohol yields increased after pulse addition of furfural to the culture. Furthermore, the maximum specific conversion rate of furfural (0.6 g/g.h) in dynamic experiments was significantly higher than what was attainable in the chemostat experiments. The dynamic furfural conversion could be described by the use of a simple Michaelis-Menten-type kinetic model. Also furfural conversion under steady-state conditions could be explained by a Michaelis-Menten-type kinetic model, but with a higher affinity and a lower maximum conversion rate. This indicated the presence of an additional component with a higher affinity, but lower maximum capacity, either in the transport system or in the conversion system of furfural.

Anaerobiosis↗

Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae.

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.

Acetoin↗

Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors.

This work describes a novel approach to detoxify lignocellulosic hydrolysates and facilitate the analysis of inhibitory compounds, namely supercritical fluid extraction (SFE). The efficiency of the fermentation of lignocellulosic dilute-acid hydrolysates depends upon the composition of the hydrolysate and the organism used. Furthermore, it has been shown that inhibitors in the hydrolysate reduce the fermentation yield. This knowledge has given rise to the need to identify and remove the inhibiting compounds. Sample clean-up or work-up steps, to provide a clean and concentrated sample for the analytical system, facilitate the characterization of inhibitors, or indeed any compound in the hydrolysates. Removal of inhibitors was performed with countercurrent flow supercritical fluid extraction of liquid hydrolysates. Three different groups of inhibitors (furan derivatives, phenolic compounds, and aliphatic acids) and sugars were subsequently analyzed in the hydrolysate, extracted hydrolysate, and extract. The effect of the SFE treatment was examined with respect to fermentability with Saccharomyces cerevisiae. Not only did the extraction provide a clean and concentrated sample (extract) for analysis, but also a hydrolysate with increased fermentability as well as lower concentrations of inhibitors such as phenolics and furan derivatives.

Bioreactors↗

Ethanol production from glucose and dilute-acid hydrolyzates by encapsulated S. cerevisiae.

The performance of encapsulated Saccharomyces cerevisiae CBS 8066 in anaerobic cultivation of glucose, in the presence and absence of furfural as well as in dilute-acid hydrolyzates, was investigated. The cultivation of encapsulated cells in 10 sequential batches in synthetic media resulted in linear increase of biomass up to 106 g/L of capsule volume, while the ethanol productivity remained constant at 5.15 (+/-0.17) g/L x h (for batches 6-10). The cells had average ethanol and glycerol yields of 0.464 and 0.056 g/g in these 10 batches. Addition of 5 g/L furfural decreased the ethanol productivity to a value of 1.31 (+/-0.10) g/L x h with the encapsulated cells, but it was stable in this range for five consecutive batches. On the other hand, the furfural decreased the ethanol yield to 0.41-0.42 g/g and increased the yield of acetic acid drastically up to 0.068 g/g. No significant lag phase was observed in any of these experiments. The encapsulated cells were also used to cultivate two different types of dilute-acid hydrolyzates. While the free cells were not able to ferment the hydrolyzates within at least 24 h, the encapsulated yeast successfully converted glucose and mannose in both of the hydrolyzates in less than 10 h with no significant lag phase. However, since the hydrolyzates were too toxic, the encapsulated cells lost their activity gradually in sequential batches.

Acids↗

Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds.

Synthetic mixtures of predominant lignocellulosic hexose sugars were supplemented with separate aliquots of three inhibitory compounds (furfural, hydroxymethylfurfural (HMF), and acetic acid) in a series of concentrations and fermented by the spent sulfite liquor (SSL)-adapted yeast strain Tembec T1 and the natural isolate Saccharomyces cerevisiae (S. cerevisiae) Y-1528 to compare tolerance and assess fermentative efficacy. The performance of Y-1528 exceeded that of Tembec T1 by a significant margin, with faster hexose sugar consumption, higher ethanol productivity, and in the case of furfural and HMF, faster inhibitor consumption. Nevertheless, furfural had a dose-proportionate effect on sugar consumption rate and ethanol productivity in both strains, but did not substantially affect ethanol yield. HMF had a similar effect on sugar consumption rate and ethanol productivity, and also lowered ethanol yield. Surprisingly, acetic acid had the least impact on sugar consumption rate and ethanol productivity, and stimulated ethanol yield at moderate concentrations. Sequential iterations of softwood (SW) and hardwood (HW) SSL were subsequently inoculated with the two yeast strains in order to compare adaptation to, and performance in lignocellulosic substrates in a cell recycle batch fermentation (CRBF) regime. Both strains were severely affected by the HW SSL, which was attributed to specific syringyl lignin-derived degradation products and synergistic interactions between inhibitors. Though ethanologenic capacity was preserved, a net loss of performance was evident from both strains, indicating the absence of adaptation to the substrates, regardless of the sequence in which the SSL types were employed.

Acetic Acid↗

[Kinetics of furfural formation as related to heating, concentration and storage of orange juices and concentrates].

According to studies on an orange concentrate diluted back with distilled water to the desired extract content, the formation of furfural follows the pattern of a consequent reaction. After the induction period, the reaction curve takes a linear course cooresponding to a reaction of a zero order. The temperature dependence of the resultant reaction constant k obeys the ARRHENIUS equation and yields the same energy of activation for all the extract contents under investigation. This is a case of specific acid catalysis, because the reaction velocity is directly proportional to the hydronium ion activity. On a semilogarithmic scale, the dependence of the k values of the extract contents of the concentrates and of the re-constituted juice can be presented for various temperatures in the form of parallels. According to the literature, the amount of the furfural produced correlates with the quality of the orange juices and concentrates. Consequently, this amount might be used as an indicator in evaluating quality deteriorations due to unsuitable manufacturing and storage on the one hand, and for the computation and optimization of the manufacturing processes and parameters on the other hand.

Food Handling↗