Acid Fermentation Research Articles

Concentrated Acid Impregnation, Hydrolysis, and Fermentation of Reed to Ethanol

The technical feasibility of bioethanol production from reed, energy crop was evaluated by way of a simple acid treatment at a low temperature. The main goal of this study was to investigate the effect of various acid-to-biomass ratios, impregnation durations, and hydrolysis periods on conversion yield of reed to monomeric sugars and inhibitors production. As a result, the maximum glucan and xylan conversion yield, 90% and 95%, respectively, was obtained at 2:1 acid-to-biomass ratio, 5-h impregnation, and 2-h hydrolysis. The extreme acidity and increase in retention time under high temperature were the main factors to increase inhibitor concentration. Fermentation of reed-derived hydrolysate with Saccharomyces cerevisiae yielded 0.45 g-ethanol/g-glucose which is lower than that of the reference fermentation (0.49 g/g) due to inhibitors; HMF, furfural, acetic acid, and formic acid.

Thermodegradation of medium-chain-length poly(3-hydroxyalkanoates) produced by Pseudomonas putida from oleic acid

Medium-chain-length poly(3-hydoxyalkanoates) (mcl-PHA), comprising six to fourteen carbon-chain-length monomers, are natural thermoplastic polyesters synthesized by fluorescent pseudomonades. In this study, mcl-PHA was produced by Pseudomonas putida from oleic acid in aerobic shake flask fermentation. Thermal degradation of mcl-PHA was performed at temperatures in the range of 160–180 °C. Thermodynamic parameters of mcl-PHA thermal degradation were determined where degradation activation energy, E d and pre-exponential factor, A equal to 85.3 kJ mol −1 and 6.07 × 10 5 s −1, respectively; and exhibited a negative activation entropy (∆ S) of −139.4 J K −1 mol −1. Titration was carried out to determine the carboxylic terminal concentration and used to correlate number-average molecular weight ( M n ) of the polymers. Thermally-degraded PHA contained higher amount of carboxylic terminals and lower M n compared to the initial PHA and these results coincide with the decreased M n in GPC analysis. Thermal properties of initial and degraded mcl-PHA were characterized by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The thermal decomposition mechanism was investigated following the analyses of the degradation products using 400-MHz 1H NMR, FTIR spectroscopy and GC analysis. The overall decomposition reaction is the hydrolysis of ester linkages to produce hydroxyl and carboxylic terminals. A small proportion of unsaturated side chain fragments would undergo oxidative cleavage at C C linkages, producing minor amount of low-molecular weight esters and acids. At higher temperatures, the hydroxyl terminal can undergo dehydration to form an alkenyl terminal.

Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae

Dicarboxylic acids that are produced from renewable resources are becoming attractive building blocks for the polymers industry. In this respect, fumaric acid is very interesting. Its low aqueous solubility facilitates product recovery. To avoid excessive waste salt production during downstream processing, a low pH for fumaric acid fermentation will be beneficial. Studying the influence of pH, working volume and shaking frequency on cell cultivation helped us to identify the best conditions to obtain appropriate pellet morphologies of a wild type strain of Rhizopus oryzae. Using these pellets, the effects of pH and CO 2 addition were studied to determine the best conditions to produce fumaric acid in batch fermentations under nitrogen-limited conditions with glucose as carbon source. Decreasing either the fermentation pH below 5 or increasing the CO 2 content of the inlet air above 10% was unfavourable for the cell-specific productivity, fumaric acid yield, and fumaric acid titer. However, switching off the pH control late in the batch phase did not affect these performance parameters and allowed achieving pH of 3.6. A concentration of 20 g L −1 of fumaric acid was obtained at pH 3.6 while the average cell mass specific productivity and fumaric acid yield were the same as at pH 5.0. Consequently, relatively modest amounts of inorganic base were required for pH control, while recovery of the acid should be relatively easy at pH 3.6.

Ensiling natural meadow forage in «Tierra del Fuego», Argentina

Livestock activity in the Argentinian «Tierra del Fuego» island is based on highly variable natural grasslands (NG). The conservation of fodder, despite the unfavorable environment, is restricted to hay; conservation as silage could be constrained by fodder quality and low temperatures. The objective was to assess the quality of NG silages fermented under contrasting storing conditions. Typical meadow forage was ensiled in minisilos and stored under shelter (Shelter) or in the field (Field) in a complete block design with repeated measures in time (t). Forage was chopped and inoculated with lactic acid bacteria plus enzymes, and harvested with 420 g dry matter (DM) kg–1 fresh matter, 111 g crude protein kg–1 DM, 665 g ash-free neutral detergent fibre kg–1 DM, and 64 g water soluble carbohydrates kg–1 DM. Shelter minisilos had higher metabolizable energy concentration at 236 d (PTrat×t = 0.03; Shelter = 10.2 ; Field = 9.6 MJ kg–1 DM, P = 0.01), and lower DM losses (Shelter = –0.2; Campo = 22%, P = 0.02) and values of pH and N-NH3/total N (Field: 5.6 and 12% and Shelter: 4.4 and 6.8%, P = 0.01). Fermentation acids concentration was similar for both treatments with preponderance of lactic acid, but acetic acid concentration increased over time (Pt = 0.01). It was concluded that in «Tierra del Fuego», natural meadow forage quality is compatible with ensiling, but environmental conditions can limit the fermentation process.

Bioethanol production from Lantanacamara (red sage): Pretreatment, saccharification and fermentation

Lantana camara contains 61.1% (w/w) holocellulose and can serve as a low-cost feedstock for bioethanol production. Acid hydrolysis (3.0%, v/v H 2SO 4, 120 °C for 45 min) of L. camara produced 187.14 mg/g total sugars along with fermentation inhibitors such as phenolics (8.2 mg/g), furfurals (5.1 mg/g) and hydroxy methyl furfurals (6.7 mg/g). Sequential application of overliming (pH 10.0) and activated charcoal (1.5%, w/v) adsorption was used to remove these toxic compounds from the acid hydrolysate. The acid-pretreated biomass of L. camara was further delignified through combined pretreatment of sodium sulphite (5.0% w/v) and sodium chlorite (3.0% w/v), which resulted in about 87.2% lignin removal. The enzymatic hydrolysis of delignified cellulosic substrate showed 80.0% saccharification after 28 h incubation at 50 °C and pH 5.0. Fermentation of acid and enzymatic hydrolysates with Pichia stipitis and Saccharomyces cerevisiae gave rise to 5.16 and 17.7 g/L of ethanol with corresponding yields of 0.32 and 0.48 g/g after 24 and 16 h, respectively.

Contaminant Removal Processes in Subsurface-Flow Constructed Wetlands: A Review

The main contaminant removal processes occurring in subsurface-flow constructed wetlands treating wastewater are reviewed. Redox conditions prevailing in the wetlands are analyzed and linked to contaminant removal mechanisms. The removal of organic matter and its accumulation in the granular medium of the wetlands are evaluated with regard to particulate and dissolved components and clogging processes. The main biological processes linked to organic matter transformation—aerobic respiration, denitrification, acid fermentation, sulfate reduction, and methanogenesis—are reviewed separately. The processes of removal of surfactants, pesticides and herbicides, emergent contaminants, nutrients, heavy metals and faecal organisms are analyzed. Advances in wetland modeling are presented as a powerful tool for understanding multiple interactions occurring in subsurface-flow constructed wetlands during the removal of contaminants.

Study on Flocculants of Lactic Acid Fermentation Broth

Flocculation of lactic acid (LA) fermentation broth using chitosan and cationic polyacrylamide (CPAM) as flocculants individually was evaluated. Flocculation effects under different flocculants dosages as well as the loss of the target product LA were investigated. It was found that CPAM was more suitable in flocculation of LA fermentation broth while the optimized dosage of CPAM was 0.2g/L. The results showed that flocculants made little impact on the LA so that it was feasible to treat lactic acid by flocculation.

Participation of phenolic acids of microbial origin in the dysfunction of mitochondria in sepsis

The role of low-molecular-weight phenolic acids of microbial origin in the mitochondrial dysfunction observed in sepsis has been studied. It was shown that microbial phenolic acids formed during fermentation of aromatic amino acids and polyphenols have an effect on mitochondrial functions, whose magnitude depends on the structure of a particular phenolic acid. The anaerobic metabolites cinnamic and benzoic acids and, to a lesser extent, phenylpropionic and phenylacetic acids at concentrations of 0.02–0.1 mM inhibited the NAD-dependent respiration, decreased the Ca2+-retention capacity of mitochondria, and oxidized the thiol groups. Their effects were partially abolished by menadione and dithiothreitol. Hydroxylated phenolic acids, 2,4-dihydroxybenzoic, 2,3-dihydroxyphenylpropionic, and other phenolic acids formed in aerobic metabolism of bacteria, when used at the same concentrations, did not affect these processes. During the catabolism of phenolic acids by clinically important bacteria, these compounds undergo anaerobic interconversions. The data obtained suggest that they contribute to the mitochondrial dysfunction in sepsis, and this contribution increases under hypoxic conditions.

Quantitative analysis and engineering of fatty acid biosynthesis in E. coli

Fatty acids are central hydrocarbon intermediates in the biosynthesis of diesel from renewable sources. We have engineered an Escherichia coli cell line that produces 4.5 g/L/day total fatty acid in a fed-batch fermentation. However, further enhancement of fatty acid biosynthesis in this cell line proved unpredictable. To develop a more reliable engineering strategy, a cell-free system was developed that enabled direct, quantitative investigation of fatty acid biosynthesis and its regulation in E. coli. Using this system, the strong dependence of fatty acid synthesis on malonyl-CoA availability and several important phenomena in fatty acid synthesis were verified. Results from this cell-free system were confirmed via the generation and analysis of metabolically engineered strains of E. coli. Our quantitative findings highlight the enormous catalytic potential of the E. coli fatty acid biosynthetic pathway, and target specific steps for protein and metabolic engineering to enhance the catalytic conversion of glucose into biodiesel.

Influence ofWilliopsis saturnusyeasts in combination withSaccharomyces cerevisiaeon wine fermentation

To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. For this study, fermentations were performed in sterilized grape must at 18 degrees C. Inoculum level was 5 x 10(6) cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml(-1). It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.

Biorefining of lignocellulosic feedstock – Technical, economic and environmental considerations

Biorefinery, an example of a multiple products system, integrates biomass conversion processes and equipment to produce fuels, power and chemicals from biomass. This study focuses on technical design, economic and environmental analysis of a lignocellulosic feedstock (LCF) biorefinery producing ethanol, succinic acid, acetic acid and electricity. As the potential worldwide demand of succinic acid and its derivatives can reach 30million tons per year, succinic acid is a promising high-value product if production cost and market price are substantially lowered. The results of the economic analysis show that the designed refinery has great potentials compared to the single-output ethanol plant; even when the price of succinic acid is lowered or the capital investment doubled. In terms of eco-efficiency, the LCF biorefinery shows better environmental performances mainly in global warming potential due to CO2 fixation during acid fermentation. The overall evaluation of the eco-efficiency depends on the importance attached to each impact category.

Effects of lactokinins from fermented acid goat whey on lipid content and adipogenesis of immortalised human adipocytes

Acid goat whey fermentation by a co-culture of Kluyveromyces marxianus and Lactobacillus rhamnosus yielded a hydrolysate inhibitory for the angiotensin-I-converting enzyme. The potential of this hydrolysate and its main lactokinin tryptophan against accumulation of lipid was investigated at different stages of immortalised human adipocytes development using Oil Red O staining and compared with that of captopril. A slight reduction of lipid content (8%) was observed when mature adipocytes were incubated with the whole hydrolysate at the concentration of 587 μg mL −1. Proliferation of pre-adipocytes was also reduced after incubation with this hydrolysate, which was not cytotoxic. Tryptophan induced a reduction of lipid content of 18% and was cytotoxic towards adipocytes at the concentration found in the hydrolysate: 25.7 μg mL −1. A decrease of the lipid content of mature adipocytes of 28% was observed with 2.9 ng mL −1 of captopril, which was also cytotoxic.

Metabolite stress and tolerance in the production of biofuels and chemicals: Gene‐expression‐based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum

Metabolite accumulation has pleiotropic, toxic, or beneficial effects on cell physiology, but such effects are not well understood at the molecular level. Cells respond and adapt to metabolite stress by mechanisms largely unexplored, especially in the context of multiple and simultaneous stresses. Solventogenic and related clostridia have an inherent advantage for production of biofuels and chemicals directly from cellulosic material and other complex carbohydrates, but issues of product/metabolite tolerance and related culture productivities remain. Using DNA microarray-based gene expression analysis, the transcriptional-stress responses of Clostridium acetobutylicum to fermentation acids acetate and butyrate and the solvent product butanol were analyzed and compared in the context of cell physiology. Ontological analysis demonstrated that stress by all three metabolites resulted in upregulation of genes related to post-translational modifications and chaperone activity, and downregulation of the translation-machinery genes. Motility genes were downregulated by acetate-stress only. The general metabolite stress included upregulation of numerous stress genes (dnaK, groES, groEL, hsp90, hsp18, clpC, and htrA), the solventogenic operon aad-ctfA-ctfB, and other solventogenic genes. Acetate stress downregulated expression of the butyryl-CoA- and butyrate-formation genes, while butyrate stress downregulated expression of acetate-formation genes. Pyrimidine-biosynthesis genes were downregulated by most stresses, but purine-biosynthesis genes were upregulated by acetate and butyrate, possibly for thiamine and histidine biosynthesis. Methionine-biosynthesis genes were upregulated by acetate stress, indicating a possibly conserved stress response mechanism also observed in Escherichia coli. Nitrogen-fixation gene expression was upregulated by acetate stress. Butyrate stress upregulated many iron-metabolism genes, riboflavin-biosynthesis genes, and several genes related to cellular repair from oxidative stress, such as perR and superoxide dismutases. Butanol stress upregulated the glycerol metabolism genes glpA and glpF. Surprisingly, metabolite stress had no apparent effect on the expression of the sporulation-cascade genes. It is argued that the list of upregulated genes in response to the three metabolite stresses includes several genes whose overexpression would likely impart tolerance, thus making the information generated in this study, a valuable source for the development of tolerant recombinant strains.

Bioethanol Production from Lignocellulosic Materials: Pretreatment, Saccharification and Fermentation

In the lignocellulosic materials (wheat stem, pearl millet stem and sorghum stem), the cellulose content is 35–38%, lignin is 12–25%, hemicelluloses is 25–30% and rest is other components. The sugar concentrate after pretreatment [Dilute sulphuric acid (2%v/v), 121°C, 15 psi for 30 min] of wheat stem (170 mg/g), pearl millet stem (120 mg/g) and sorghum stem (lOOmg/g). Sequential application of overtiming (pH 10.0) and activated charcoal (1.5%, w/v) adsorption was used to remove these toxic compounds from the acid hydrolysate. The acid-pre-treated biomass of wheat stem, pearl millet stem and sorghum stem was further delignified through combined pretreatment of sodium sulphite (5.0% w/v) and sodium chlorite (3.0% w/v), which resulted in about 67 - 75% lignin removal. Enzymatic hydrolysis with combinations of enzymes of delignified cellulosic substrates showed best saccharification after 78 h incubation at 500°C and pH 5.0 in pearl millet (95%). Fermentation of acid and enzymatic hydrolysates with Candida tropicalis and saccharomyces cere-visiae at 30°C and pH 6.0, wheat stem 16.4 g/l, pearl millet 31.4 g/l and 16.3g/l of ethanol concentration with corresponding yields of 0.39, 0.37and 0.41g/g after 78 h, respectively.

Strain improvement of Lactobacillus lactis for d-lactic acid production

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased D: -lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest D: -lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into D-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain D-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.

Rapid monitoring of mRNA levels with a molecular beacon during microbial fermentation

In the microbial fermentation bioreactor, the processes of mRNA transcription, protein translation, and enzyme-catalyzed biosynthesis remain as “black boxes” of industrial monitoring and process control. Monitoring the kinetics of these “black boxes” is very helpful for optimizing and controlling the microbial fermentation process. This study first applied a molecular beacon (MB) to monitor the changes in the mRNA level of the phzC gene during antibiotic phenazine-1-carboxylic acid fermentation. Seven typical MB hybridization buffers were compared, and the effect of formamide on MBs was also studied. The results showed that rapid monitoring of the mRNA level using MBs was feasible. The optimal hybridization buffer for phzC MB was 100 mM Tris, 1 mM MgCl 2, pH 8.0. The optimal hybridization temperature was 35 °C, and formamide proved unsuitable for MB hybridization. The limit of detection of phzC MB was 1.67 nM and MB hybridization was complete by 7 min. Given that the time for RNA extraction is 12 min, it is possible that monitoring of phzC mRNA can be completed in less than 20 min. Since production of most amine acids, organic acids, wines, antibiotics, and proteins relies on microbial fermentation, our method may have some potential for application in these other microbial industries.

Effect of Ensilaging and Organic Solvent Treatment on Activity of Proteases from Chicken Intestine

Studies were carried out to assess the extractability of proteins from chicken intestine and to determine the activity of proteases in crude extract. Further, the activity of proteases was evaluated in acid and fermented chicken intestinal silage as well as in organic solvent-treated intestinal samples. Extractability of proteins was in the range of 3.85–4.06 mg/ml and was not affected (p ≥ 0.05) by the four different extractants, viz., distilled water, phosphate buffer, and 1% potassium chloride with or without 0.1 M Na2EDTA. The specific activity of acidic and alkaline proteases were in the range of 4.78–7.26 and 19.31–24.07 units, respectively, and was not affected (p ≥ 0.05) by the type of extractant used. The pH profile for activity showed higher activity in the alkaline pH range. Addition of acid or ingredients for fermentation reduced the extractability of protein significantly (p ≤ 0.05). During storage of silage, the reduction in enzyme activity was higher in acid silage compared with fermented silage. Treatment of chicken intestine with organic solvents enhanced the enzyme activity and higher increase was observed with isopropyl alcohol-treated intestine. During storage of solvent-treated powders up to 3 months, the activity of proteases reduced in all the solvent-treated samples highest being in acetone-treated powder.

Utilizing RNA/DNA hybridization to directly quantify mRNA levels in microbial fermentation samples

mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly quantify mRNA from fermentation samples. After total RNA was extracted, the mRNA was hybridized and protected by a longer DNA oligonucleotide. The probe length determined the strength of signal amplification. S1 nuclease and RNase A were used to remove excess probe, single-stranded RNA, and mis-matched RNA/DNA hybrids. Finally, the perfect-matched RNA/DNA hybrid was quantified by SYBR Green I dye. The conditions of liquid hybridization and enzyme digestion were systemically optimized. The kinetic tendency of phzC mRNA levels during phenazine-1-carboxylic acid fermentation was consistent with the results from MB hybridization in our previous report. The detection of mRNA levels of ten genes in Pseudomonas sp. M18G proved that this method is universal and feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.

Propionic acid production from glycerol by metabolically engineered Propionibacterium acidipropionici

Large amounts of crude glycerol produced in the biodiesel industry can be used as a low-cost renewable feedstock to produce chemicals and fuels. Compared to sugars (sucrose, glucose, xylose, etc.), glycerol has a lower reducing level, which is of benefit to the production of reduced chemicals. In this work, glycerol as the sole carbon source in propionic acid fermentation by metabolically engineered Propionibacterium acidipropionici (ACK-Tet) was studied. It was found that the adapted ACK-Tet mutant could use glycerol for its growth and produced propionic acid at a high yield of 0.54–0.71 g/g, which was much higher than that from glucose (∼0.35 g/g). In addition, the production of acetic acid in glycerol fermentation was much less than that from glucose. Thus, glycerol fermentation produced a high purity propionic acid with a high propionic acid to acetic acid ratio of 22.4 (vs. ∼5 for glucose fermentation), facilitating the recovery and purification of propionic acid from the fermentation broth. The highest propionic acid concentration obtained from glycerol fermentation was ∼106 g/L, which was 2.5 times of the highest concentration (∼42 g/L) previously reported in the literature.

Dilution rates influence ammonia-assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture

The objective of this study was to examine the effect of dilution rates (Ds, varying from 0.05 to 0.42 h(-1)) in glucose-limited continuous culture on cell yield, cell composition, fermentation pattern and ammonia assimilation enzymes of Selenomonas ruminantium strain D. All glucose-limited continuous culture experiments were conducted under anaerobic conditions. Except for protein, all cell constituents including carbohydrates, RNA and DNA yielded significant cubic responses to Ds with the highest values at Ds of either 0.10 or 0.20 h(-1). At Ds higher than 0.2 h(-1), fermentation acid pattern shifted primarily from propionate and acetate to lactate production. Succinate also accumulated at the higher Ds (0.30 and 0.42 h(-1)). Glucose was most efficiently utilized by S. ruminantium D at 0.20 h(-1) after which decreases in glucose and ATP yields were observed. Under energy limiting conditions, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) appeared to be the major enzymes involved in nitrogen assimilation suggesting that other potential ammonia incorporating enzymes were of little importance in ammonia assimilation in S. ruminantium D. GS exhibited lower activities than GDH at all Ds, which indicates that the bacterial growth rate is not a primary regulator of their activities. Studied dilution rates influenced cell composition, fermentation pattern and nitrogen assimilation of S. ruminantium strain D grown in glucose-limited continuous culture. Selenomonas ruminantium D is an ecologically and evolutionary important bacterium in ruminants and is present under most rumen dietary conditions. Characterizing the growth physiology and ammonia assimilation enzymes of S. ruminantium D during glucose limitation at Ds, which simulate the liquid turnover rates in rumen, will provide a better understanding of how this micro-organism responds to differing growth conditions.