Effects of acetate on the growth and fermentation performance of Escherichia coli KO11

Escherichia coli KO11, in which the genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) encoding the ethanolpathway from Zymomonas mobili were inserted into the chromosome, has been shown to metabolize all major sugars that are consituents of hemicellulosic hydrolysates to ethanol, in anaerobic conditions. However, the growth and fermentation performance of this recombinant bacteria may be affected by acetic acid a potential inhibitor present in hemicellulose hydrolysates in a range of 2.0–15.0 g/L. It was observed that acetate affected the growth of E. coli KO11, prolonging the lag phase and inducing loss of biomass production and reduction of growth rate. At lower pH levels, the sensitivity to acetic acid was enhanced owing to the increased concentration of the protonated species. On the other hand, the recombinant bacteria showed a high tolerance to acetic acid regarding fermentative performance. In Luria broth medium with glucose or xylose as a single sugar source, it was observed that neither yield nor productivity was affected by the addition of acetate in a range of 2.0–12.0 g/L, suggesting some uncoupling of the growth vs ethanol production.     

Xylitol production from hardwood hemicellulose hydrolysates by Pachysolen tannophilus, Debaryomyces hansenii, and Candida guilliermondii

Three different yeasts, Pachysolen tannophilus, Debaryomyces hansenii, and Candida guilliermondii, were evaluated to ferment xylose solutions prepared from hardwood hemicellulose hydrolysates, among which P. tannophilus proved to be the most promising microorganism. However, the presence of both lignin-derived compounds (LDC) and acetic acid rendered a poor fermentation. To enhance the fermentation kinetics, different treatments to purify the hydrolysates were studied, including overliming, charcoal adsorption for LDC removal, and evaporation for acetic acid and furfural stripping. Under the best operating conditions assayed, 39.5g/L of xylitol were achieved after 96 h of fermentation, which corresponds to a volumetric productivity of 0.41 g/L·h and a yield of product on consumed substrate of 0.63 g _p /g_S.     

Influence of inhibitory compounds and minor sugars on xylitol production by <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis>

To obtain in-depth information on the overall metabolic behavior of the new good xylitol producer Debaryomyces hansenii UFV-170, batch bioconversions were carried out using semisynthetic media with compositions simulating those of typical acidic hemicellulose hydrolysates of sugarcane bagasse. For this purpose, we used media containing glucose (4.3–6.5 g/L), xylose (60.1–92.1 g/L), or arabinose (5.9–9.2 g/L), or binary or ternary mixtures of them in either the presence or absence of typical inhibitors of acidic hydrolysates, such as furfural (1.0–5.0 g/L), hydroxymethylfurfural (0.01–0.30 g/L), acetic acid (0.5–3.0 g/L), and vanillin (0.5–3.0 g/L). D. hansenii exhibited a good tolerance to high sugar concentrations as well as to the presence of inhibiting compounds in the fermentation media. It was able to produce xylitol only from xylose, arabitol from arabinose, and no glucitol from glucose. Arabinose metabolization was incomplete, while ethanol was mainly produced from glucose and, to a lesser less extent, from xylose and arabinose. The results suggest potential application of this strain in xyloseto-xylitol bioconversion from complex xylose media from lignocellulosic materials.     

Fermentation of sugars in organe peel hydrolysates to ethanol by recombinantEscherichia coli KO11

The conversion of monosaccharides in organe peel hydrolysates to ethanol by recombinantEscherichia coli KO11 has been investigated in pH-controlled batch fermentations at 32 and 37°C. pH values and concentration of peel hydrolysate were varied to determine approximate optimal conditions and limitations of these fermentations. Very high yields of ethanol were achieved by this microorganism at reasonable ethanol concentrations (28–48 g/L). The pH range between 5.8 and 6.2 appears to be optimal. The microorganism can convert all major monosaccharides in organe peel hydrolysates to ethanol and to smaller amounts of acetic and lactic acids. Acetic acid is coproduced in equimolar amounts with ethanol by catabolism of salts of galacturonic acid. South Atlantic Area, Agricultural Research Service, US Department of Agriculture. Mention of a trademark or proprietary product is for identification only, and does not imply a guarantee or warranty of the product by the US Department of Agriculture. All programs and services of the US Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

Enhancement of acid tolerance in Zymomonas mobilis by a proton-buffering peptide

A portion of the cbpA gene from Escherichia coli K-12 encoding a 24 amino acid proton-buffering peptide (Pbp) was cloned via the shuttle vector pJB99 into E. coli JM105 and subsequently into Zymomonas mobilis CP4. Expression of Pbp was confirmed in both JM105 and CP4 by HPLC. Z. mobilis CP4 carrying pJB99-2 (Pbp) exhibited increased acid tolerance (p<0.05) in acidified TSB (HCl [pH 3.0] or acetic acid [pH 3.5]), glycine-HCl buffer (pH 3.0), and sodium acetate-acetic acid buffer (pH 3.5) in comparison to the parent strain (CP4) and CP4 with pJB99 (control plasmid). Although the expression of Pbp influenced survival at a low pH, the minimum growth pH was unaffected. Growth of Z. mobilis in the presence of ampicillin also significantly increased acid tolerance by an unknown mechanism. Results from this study demonstrate that the production of a peptide with a high proportion of basic amino acids can contribute to protection from low pH and weak organic acids such as acetic acid.     

The combined effects of acetic acid, formic acid, and hydroquinone on Debaryomyces hansenii physiology

The combined effects of inhibitors present in lignocellulosic hydrolysates was studied using a multivariate statistical approach. Acetic acid (0–6 g/L), formic acid (0–4.6 g/L) and hydroquinone (0–3 g/L) were tested as model inhibitors in synthetic media containing a mixture of glucose, xylose, and arabinose simulating concentrated hemicellulosic hydrolysates. Inhibitors were consumed sequentially (acetic acid, formic acid, and hydroquinone), alongside to the monosaccharides (glucose, xylose, and arabinose). Xylitol was always the main metabolic product. Additionally, glycerol, ethanol, and arabitol were also obtained. The inhibitory action of acetic acid on growth, on glucose consumption and on all product formation rates was found to be significant (p≤0.05), as well as formic acid inhibition on xylose consumption and biomass production. Hydroquinone negatively affected biomass productivity and yield, but it significantly increased xylose consumption and xylitol productivity. Hydroquinone interactions, either with acetic or formic acid or with both, are also statistically signficant. Hydroquinone seems to partially lessen the acetic acid and amplify formic acid effects. The results clearly indicate that the interaction effects play an important role on the xylitol bioprocess.     

Growth Inhibition of Thermotolerant Yeast,Kluyveromyces marxianus,in Hydrolysates from Cassava Pulp

In this study, we report the inhibition of Kluyveromyces marxianus TISTR5925 growth and ethanol fermentation in the presence of furan derivatives and weak acids (acetic acid and lactic acid) at high temperatures. Cassava pulp, obtained as the waste from starch processing, was collected from 14 starch factories located in several provinces of Thailand. At a high temperature (42 °C), the cassava pulp hydrolysate from some starch factories strongly inhibited growth and ethanol production of both K. marxianus (strain TISTR5925) and Saccharomyces cerevisiae (strain K3). HPLC detected high levels of lactic acid and acetic acid in the hydrolysates, suggesting that these weak acids impaired the growth of K. marxianus at high temperature. We isolated Trp-requiring mutants that had reduced tolerance to acetic acid compared to the wild-type. This sensitivity to acetic acid was suppressed by supplementation of the medium with tryptophan.     

Effects of aliphatic acids, furfural, and phenolic compounds on Debaryomyces hansenii CCMI 941

Debaryomyces hansenii is a polyol overproducing yeast that can have a potential use for upgrading lignocellulosic hydrolysates. Therefore, the establishment of its tolerance to metabolic inhibitors found in hydrolysates is of major interest. We studied the effects of selected aliphatic acids, phenolic compounds, and furfural. Acetic acid favored biomass production for concentrations <6.0 g/L. Formic acid was more toxic than acetic acid and induced xylitol accumulation (maximum yield of 0.21 g/g of xylose). All tested phenolics strongly decreased the specific growth rate. Increased toxicity was found for hydroquinone, syringaldehyde, and 4-methylcatechol and was correlated to the compound’s hydrophobicity. Increasing the amount of furfural led to longer lag phases and had a detrimental effect on specific growth rate and biomass productivity.     

Thermophilic ethanol production investigation of ethanol yield and tolerance in continuous culture

Ethanol yield and ethanol tolerance, the two factors that most constrain the utilization of thermophilic bacteria for ethanol production, were investigated in continuous xylose-grown cultures ofClostridium thermosaccharolyticum. Under xylose-limiting conditions, including varying dilution rates and feed concentrations, the ethanol selectivity (Se, mol/mol) relative to acetic acid, lactic acid, and propane diol remained relatively constant at about 2. Product addition and removal experiments indicate that mass action effects related to the concentrations of organic fermentation products play a relatively minor role in determining the ratios of products made. Of much greater apparent importance were as yet uncharacterized regulatory mechanisms that appear to be correlated with nonlimiting concentrations of the carbon and energy-source. Substrate-plentiful transients were found to accompany Se values > 11. Such transients provide a useful model system for the study of end product control, as well as a cultivation mode with considerable applied potential. No apparent ethanol inhibition was observed, as indicated by no decrease in the maximum rate of growth allowing complete substrate utilization (0.22 h^-1) for endogenously-produced ethanol concentrations up to 11.4 g/L, and total endogenously-produced + exogenously-added ethanol concentrations up to 21.3 g/L. Higher concentrations of ethanol are tolerated atµ = 0.11 h^-1, although the onset of inhibition was not characterized at this growth rate. Results suggest that the ethanol tolerance of C.thermosaccharolyticum grown in continuous culture may be greater than that typically observed previously for thermophiles grown in batch culture.

     

A new reductimetric reagent: Iron (II) in acetic acid medium and in presence of pyrophosphate. Spectrophotometric titration of some oxazine dyes

Iron(II) in acetic acid medium and the presence of pyrophosphate is used as a new reductimetric reagent and utilised for the spectrophotometric titration of microgram quantities of six oxazine dyes. All these dyes are rapidly and quantitatively reduced to their colourless leuco-bases in a 2-electron reduction with iron(II), provided the medium contains 1 or 3M acetic acid (depending on the dye) and at least 0.1M pyrophosphate. The redox potentials of the iron(III)/iron(II) couple at different pyrophosphate and acetic acid concentrations have been measured and a method for purification of some of the commercially impure oxazine dyes is suggested.     

Production of propionic acid using a xylose utilizingPropionibacterium

The kinetics ofP. acidipropionici (ATCC25562), a xylose-utilizing rumen microorganism, was studied to assess its use for propionic acid production from wood hydrolyzates. Propionic acid has been shown to have a stronger inhibitory effect than acetic acid, with the undissociated acid form being responsible for the majority of the inhibitory effect. Thus, in batch tests with pH controlled at 6.0, the propionic acid concentration reaches 25 g/L and the acetic acid 7 g/L. Xylose uptake rate is dependent on the specific growth rate and glucose concentration. An immobilized cell columnar reactor at very high product yields (80%) proved adequate for propionic production. At cell concentrations of 95 g/L with high product concentration, volumetric productivities of 2.7 g/L·h were obtained in ultrafiltration cell recycle systems.     

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200–250°C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4c/gal of ethanol. This work examined the growth and fermentation performance of xyloseutilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated asthe “adapted” strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30h, followed by CP4:pZB5 at 48h, with corresponding volumetric productivities of 1.4 and 0.89 g/(L·h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to fermentarabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.     

Modeling of the hydrolysis of sugar cane bagasse with hydrochloric acid

Sugar cane bagasse was hydrolyzed under different concentrations of hydrochloric acid (2–6%), reaction times (0–300 min), and temperatures (100–128°C). Sugars obtained (xylose, glucose, arabinose, and glucose) and deg-radation products (furfural and acetic acid) were determined. Based on the Saeman model and the two-fraction model, kinetic parameters for predicting these compounds in the hydrolysates were developed. The influence of temperature was studied using the Arrhenius equation. The optimal conditions selected were 128°C, 2% HCl, and 51.1 min. Using these conditions, 22.6g xylose/L, 3.31 garabinose/L, 3.77 g glucose/L, 3.59 g acetic acid/L, and 1.54 g furfural/L were obtained.     

Cellulase production by Trichoderma reesei using sawdust hydrolysate

Sawdust hydrolysates were investigated for their ability to support cell growth and cellulase production, and for potential inhibition of Trichoderma reesei Rut C30. Simultaneous fermentations were conducted to compare the hydrolysate-based media with the controls having equivalent concentrations of glucose and Avicel cellulose. Six hydrolysates differing in the boiling durations in the hydrolysis procedure were evaluated. The hydrolysates were found to support cell growth and induce active cellulase synthesis. The maximum specific cellulase production rate was 0.046 filter paper units (FPU)/(g of cells · h) in the hydrolysate-based systems, much higher than that (0.017 FPU/[g of cells · h]) in the controls.     

Inhibition of microbial xylitol production by acetic acid and its relation with fermentative parameters

Precipitated sugarcane bagasse hemicellulosic hydrolysate containing acetic acid was fermented by Candida guilliermondii FTI 20037 under different operational conditions (pH 4.0 and 7.0, three aeration rates). At pH 7.0 and k _L a of 10 (0.75 vvm) and 22.5/h (3.0 vvm) the acetic acid had not been consumed until the end of the fermentations, whereas at the same pH and k _L a of 35/h (4.5 vvm) the acid was rapidly consumed and acetic acid inhibition was not important. On the other hand, fermentations at an initial pH of 4.0 and k _L a of 22.5 and 35/h required less time for the acid uptake than fermentations at k _L a of 10/h. The acetic acid assimilation by the yeast indicates the ability of this strain to ferment in partially detoxified medium, making possible the utilization of the sugarcane bagasse hydrolysate in this bioprocess. The effects on xylitol yield and production are reported.     

Hydrogen Bonding of Carboxylic Acids in Aqueous Solutions—UV Spectroscopy, Viscosity, and Molecular Simulation of Acetic Acid

The UV spectra of aqueous acetic acid solutions up to 2M were investigated. At these wavelengths, the carboxylic acids exhibit an absorption peak, attributed to the C=O group, which shifts when hydrogen bonds are formed.. The measured spectra were best fitted to several bands, either of Gaussian or Lorentzian shape, which can be explained as several types of structural units formed by hydrogen bonds established between acetic acid and water molecules and between acetic acid molecules themselves. Molecular dynamics simulation of these mixtures was also performed, confirming the occurrence of several types of hydrogen bonds and showing the presence of dimers at higher concentrations. The viscosity and density of these solutions were also measured at different concentrations and temperatures. These results give a more complete picture of the hydrogen bond network of the system.     

Preparation of 1-butyl-3-methylimidazolium dodecatungstophosphate and its catalytic performance for esterification of ethanol and acetic acid

1-Butyl-3-methylimidazolium dodecatungstophosphate catalyst ([bmim]₃PW₁₂O₄₀) with high water tolerance was prepared from 1-butyl-3-methylimidazolium bromide ([bmim]Br) and phosphotungstic acid (H₃PW₁₂O₄₀). The catalyst was characterized by means of Fourier transform infrared spectroscopy, thermogravimetry-differential scanning calorimetry, n-BuNH₂ potentiometric titration, elemental analysis and so on. Its catalytic activity for esterification of ethanol and acetic acid to ethyl acetate was measured. The results show that there were three crystal-water molecules in the [bmim]₃PW₁₂O₄₀ catalyst, and it preserved the primary Keggin structure and acid strength of H₃PW₁₂O₄₀. The acid amount of [bmim]₃PW₁₂O₄₀ catalyst was less than that of H₃PW₁₂O₄₀. The [bmim]₃PW₁₂O₄₀ catalyst exhibited higher catalytic activity and reusability in the esterification of ethanol and acetic acid to ethyl acetate. __________ Translated from Chinese Journal of Catalysis, 2008, 29(7) (in Chinese)     

Comparison between Different Hydrolysis Processes of Vine-Trimming Waste to Obtain Hemicellulosic Sugars for Further Lactic Acid Conversion

Trimming vine shoot samples were treated with water under selected operational conditions (autohydrolysis reaction) to obtain a liquid phase containing hemicellulose-decomposition products. In a further acid-catalyzed step (posthydrolysis reaction), xylooligosaccharides were converted into single sugars for the biotechnological production of lactic acid using Lactobacillus pentosus. A wide range of temperatures, reaction times, and acid concentrations were tested during the autohydrolysis–posthydrolysis process to investigate their influence on hemicellulose solubilization and reaction products. The maximum concentration of hemicellulosic sugars was achieved using autohydrolysis at 210 °C followed by posthydrolysis with 1% H₂SO₄ during 2 h. Data from autohydrolysis–posthydrolysis were compared with the results obtained at the optima conditions assayed for prehydrolysis (3% H₂SO₄ at 130 °C during 15 min) based on previous works. Prehydrolysis extracted more hemicellulosic sugars from trimming vine shoots; however, the protein content in the hydrolysates from autohydrolysis–posthydrolysis was higher. The harsher conditions assayed during the autohydrolysis process and the higher content of protein after this treatment could induce Maillard reactions decreasing consequently the concentration of hemicellulosic sugars in the hydrolysates. Therefore, despite the several advantages of autohydrolysis (less equipment caused by the absence of mineral acid, less generation of neutralized sludges, and low cost of reagents) the poor results obtained in this work with no detoxified hydrolysates (Q _P = 0.36 g/L h, Q _S = 0.79 g/L h, Y _P/S = 0.45 g/g, Y _P/Sth = 61.5 %) or charcoal-treated hydrolysates (Q _P = 0.76 g/L h, Q _S = 1.47 g/L h, Y _P/S = 0.52 g/g, Y _P/Sth = 71.5 %) suggest that prehydrolysis of trimming vine shoots with diluted H₂SO₄ is more attractive than autohydrolysis-posthydrolysis for obtaining lactic acid through fermentation of hemicellulosic sugars with L. pentosus. Besides the higher hemicellulosic sugars concentration achieved when using the prehydrolysis technology, no detoxification steps are required to produce efficiently lactic acid (Q _P = 1.14 g/L h; Q _S = 1.64 g/L h; Y _P/S = 0.70 g/g; Y _P/Sth = 92.6 %), even when vinification lees are used as nutrients (Q _P = 0.89 g/L h; Q _S = 1.54 g/L h; Y _P/S = 0.58 g/g; Y _P/Sth = 76.1 %).     

Fermentation of Reactive-Membrane-Extracted and Ammonium-Hydroxide-Conditioned Dilute-Acid-Pretreated Corn Stover

Acid-pretreated biomass contains various compounds (acetic acid, etc.) that are inhibitory to fermentative microorganisms. Removing or deactivating these compounds using detoxification methods such as overliming or ammonium hydroxide conditioning (AHC) improves sugar-to-ethanol yields. In this study, we treated the liquor fraction of dilute-acid-pretreated corn stover using AHC and a new reactive membrane extraction technique, both separately and in combination, and then the sugars in the treated liquors were fermented to ethanol with the glucose–xylose-fermenting bacterium, Zymomonas mobilis 8b. We performed reactive extraction with mixtures of octanol/Alamine 336 or oleyl alcohol/Alamine 336. The best ethanol yields and rates were achieved for oleyl alcohol-extracted hydrolysates followed by AHC hydrolysates, while octanol-extracted hydrolysates were unfermentable because highly toxic octanol was found in the hydrolysate. Adding olive oil significantly improved yields for octanol-extracted hydrolysate. Additional work is underway to determine if this technology is a cost-effective alternative to traditional hydrolysate conditioning processes.     

Effect of acetic acid and furfural on cellulase production of Trichoderma reesei RUT C30

Because of the high temperature applied in the steam pretreatment of lignocellulosic materials, different types of inhibiting degradation products of saccharides and lignin, such as acetic acid and furfural, are formed. The main objective of the present study was to examine the effect of acetic acid and furfural on the cellulase production of a filamentous fungus Trichoderma reesei RUT C30, which is known to be one of the best cellulase-producing strains. Mandels’s mineral medium, supplemented with steam-pretreated willow as the carbon source at a concentration corresponding to 10 g/L of carbohydrate, was used. Four different concentration levels of acetic acid (0–3.0 g/L) and furfural (0–1.2 g/L) were applied alone as well as in certain combinations. Two enzyme activities, cellulase and β-glucosidase, were measured. The highest cellulase activity obtained after a 7-d incubation was 1.55 FPU/mL with 1.0 g/L of acetic acid and 0.8 g/L of furfural added to the medium. This was 17% higher than that obtained without acetic acid and furfural. Furthermore, the results showed that acetic acid alone did not influence the cellulase activity even at the highest concentration. However, β-glucosidase activity was increased with increasing acetic acid concentration. Furfural proved to be an inhibiting agent causing a significant decrease in both cellulase and β-glucosidase production.