Continuous production of lactic acid in a cell recycle reactor

The production of lactic acid from glucose has been demonstrated using a CSTR (continuous stirred-tank reactor) with cell recycle. Studies were conducted withLactobacillus delbrueckii at a fermentation temperature of 42°C and a pH of 6.25. A cell density of 140 g dry weight/L and a volumetric productivity of 150 g/L.h, with complete glucose consumption, were obtained. It was not possible to obtain a lactic acid concentration above 60 g/L because of product inhibition. A cell purge was not necessary to maintain high viability bacteria culture or to obtain a steady state. At steady state the net cell growth appeared to be negligible. The specific glucose consumption for cell maintenance was 0.33 g glucose/g cells-h.     

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.     

Optimization of seed production for a simultaneous saccharification cofermentation biomass-to-ethanol process using recombinantZymomonas

The five-carbon sugard-xylose is a major component of hemicellulose and accounts for roughly one-third of the carbohydrate content of many lignocellulosic materials. The efficient fermentation of xylose-rich hemicellulose hydrolyzates (prehydrolyzates) represents an opportunity to improve significantly the economics of large-scale fuel ethanol production from lignocellulosic feedstocks. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process for ethanol production from biomass that uses a dilute-acid pretreatment and a metabolically engineered strain ofZymomonas mobilis that can coferment glucose and xylose. The objective of this study was to establish optimal conditions for cost-effective seed production that are compatible with the SSCF process design. Two-level and three-level full factorial experimental designs were employed to characterize efficiently the growth performance of recombinantZ. mobilis CP4:pZB5 as a function of nutrient level, pH, and acetic acid concentration using a synthetic hardwood hemicellulose hydrolyzate containing 4% (w/v) xylose and 0.8% (w/v) glucose. Fermentations were run batchwise and were pH-controlled at low levels of clarified corn steep liquor (cCSL, 1-2% v/v), which were used as the sole source of nutrients. For the purpose of assessing comparative fermentation performance, seed production was also carried out using a “benchmark” yeast extract-based laboratory medium. Analysis of variance (ANOVA) of experimental results was performed to determine the main effects and possible interactive effects of nutrient (cCSL) level, pH, and acetic acid concentration on the rate of xylose utilization and the extent of cell mass production. Results indicate that the concentration of acetic acid is the most significant limiting factor for the xylose utilization rate and the extent of cell mass production; nutrient level and pH exerted weaker, but statistically significant effects. At pH 6.0, in the absence of acetic acid, the final cell mass concentration was 1.4 g dry cell mass/L (g DCM/L), but decreased to 0.92 and 0.64 g DCM/L in the presence of 0.5 and 1.0% (w/v) acetic acid, respectively. At concentrations of acetic acid of 0.75 (w/v) or lower, fermentation was complete within 1.5 d. In contrast, in the presence of 1.0% (w/v) acetic acid, 25% of the xylose remained after 2 d. At a volumetric supplementation level of 1.5–2.0% (v/v), cCSL proved to be a cost-effective single-source nutritional adjunct that can support growth and fermentation performance at levels comparable to those achieved using the expensive yeast extract-based laboratory reference medium.     

Optimization of L-Lactic acid production from glucose by Rhizopus oryzae ATCC 52311

The effect of nutrients on L(+)-lactic acid production from glucose was investigated using Rhizopus oryzae ATCC 523 11. From the shake-flask experiments, the optimal medium composition was defined for improved lactic-acid production. In order to enhance lactic-acid production rate and product yield, controlled aeration in a bubble column was conducted under optimal conditions. Results showed a maximum lactic-acid production rate of 2.58 g/L/h was obtained with an initial glucose concentration of 94 g/L. Finallactic-acid concentration of 83 g/L was achieved after 32 h of fermentation with a weight of 0.88 glactic acid/g glucose consumed.     

Carob pod: A new substrate for citric acid production by Aspergillus niger

The production of citric acid from carob pod extract byA. niger in surface fermentation was investigated. A maximum citric acid concentration (85.5 g/L), citric acid productivity (4.07 g/L/d), specific citric acid production rate (0.18 g/g/d), and specific sugar uptake rate (0.358 g/g/d) was achieved at an initial sugar concentration of 200 g/L, pH of 6.5, and a temperature of 30°C. Other kinetic parameters, namely, citric acid yield, biomass yield, specific biomass production rate, and fermentation efficiency were maximum at pH 6.5, temperature 30°C, and initial sugar concentration 100 g/L. The external addition of methanol into the carob pod extract at a concentration up to 4% (v/v) improved the production of citric acid.     

Fermentation metabolism and kinetics in the production of organic acids byPropionibacterium acidipropionici

The genusPropionibacterium acidipropionici was grown under pH-controlled batch fermentation conditions for the production of acetic and propionic acids using 19.1 g/L glucose as a carbon source. The optimum pH range was found to be between 5.5 and 6.5. Bacterial metabolism and fermentation pathways were altered at pH values outside this range. Lactic acid was produced as a key intermediate, with the final acetic and propionic acid production entirely dependent on the cell's ability to metabolize the lactic acid. Most of the glucose in the medium was consumed in less than 20 h of fermentation and converted to lactic acid. Batch fermentation at pH 6 showed that lactic acid was completely utilized to produce 8.5 g/L propionic acid and 5.7 g/L acetic acid. However, the bacteria were unable to metabolize lactic acid at pH 7, resulting in 0.7 g/L propionic acid and 7.0 g/L acetic acid in the fermenter. A kinetic study of batch fermentation at pH 6 showed two distinct growth phases during the fermentation. Most of the cell growth was achieved in the exponential growth stage when glucose was consumed as a main substrate. A nonexponential growth stage was observed when lactic acid was utilized as a carbon source, producing propionic and acetic acids as secondary metabolites.     

Catalytic carbonylation of ethylene in the presence of the Pd(acac)2-m-Ph2PC6H4SO3Na(H)-AcOH system

Catalytic systems based on phosphine complexes of palladium have been developed for synthesizing propionic acid (monocarbonylation) and alternating (11) ethylene-carbon monoxide copolymers,i.e., polyketones (polycarbonylation).m-(Diphenylphosphino)benzenesulfonic acid or its sodium salt were used as ligands. Monocarbonylation proceeds at atmospheric pressure in dioxane or acetic acid solvents. Under high pressure, the reaction pathway can change from monocarbonylation, which occurs in the presense of the sodium salt of the ligand, to polycarbonylation when the sodium ion at the sulfo group is completely replaced by a proton. This change in reaction selectivity is observed when the process is performed in acetic acid. When the ligand is present both in the acid and the neutral form, products of di- and oligocarbonylation are formed along with propionic acid and the polyketone. These products were characterized by¹H and¹³C NMR spectra as alternating keto acids C₂H₅(COCH₂CH₂) _n COOH, wheren=1÷3. Kinetic equations were derived for the selective synthesis of propionic acid and polyketones.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 401–404, March, 1994.The authors are grateful to V. A. Zavel'skii who recorded the 1H NMR spectra on a Bruker CXP-200 spectrometer.     

Use of Propionibacterium freudenreichii T82 Strain for Effective Biosynthesis of Propionic Acid and Trehalose in a Medium with Apple Pomace Extract and Potato Wastewater

Propionic acid bacteria are the source of many metabolites, e.g., propionic acid and trehalose. Compared to microbiological synthesis, the production of these metabolites by petrochemical means or enzymatic conversion is more profitable. The components of microbiological media account for a large part of the costs associated with propionic fermentation, due to the high nutritional requirements of Propionibacterium. This problem can be overcome by formulating a medium based on the by-products of technological processes, which can act as nutritional sources and at the same time replace expensive laboratory preparations (e.g., peptone and yeast extract). The metabolic activity of P. freudenreichii was investigated in two different breeding environments: in a medium containing peptone, yeast extract, and biotin, and in a waste-based medium consisting of only apple pomace and potato wastewater. The highest production of propionic acid amounting to 14.54 g/L was obtained in the medium containing apple pomace and pure laboratory supplements with a yield of 0.44 g/g. Importantly, the acid production parameters in the waste medium reached almost the same level (12.71 g/L, 0.42 g/g) as the medium containing pure supplements. Acetic acid synthesis was more efficient in the waste medium; it was also characterized by a higher level of accumulated trehalose (59.8 mg/g d.s.). Thus, the obtained results show that P. freudenreichii bacteria exhibited relatively high metabolic activity in an environment with apple pomace used as a carbon source and potato wastewater used as a nitrogen source. This method of propioniate production could be cheaper and more sustainable than the chemical manner.     

Production of l-malic acid via biocatalysis employing wild-type and respiratory-deficient yeasts

Succinic acid was produced efficiently from fumaric acid by a recombinantE. coli strain DH5αt/pGC1002 containing multicopy fumarate reductase genes. The effects of initial fumaric acid and glucose concentration on the production of succinic acid were investigated. Succinic acid reached 41 to over 60 g/L in 48.5 h starting with 50 to 64 g/L fumaric acid. Significant substrate inhibition was observed at initial fumaric acid concentration of 90 g/L. L-Malic acid became the major fermentation product under these conditions. Provision of glucose (5-30 g/L) to the fermentation medium stimulated the initial succinic acid production rate over two folds.     

On-line dialysis of organic acids from a Propionibacterium freudenreichii fermentation

The efficiency of an ion exchange system coupled to a bioreactor to extract on-line inhibitory organic acids produced was evaluated. Batch fermentations without dialysis and fed-batch experiments with and without dialysis of Propio nibacterium freudenreichii were conducted. It was possible to keep the propionic acid concentration in the reactor at a low level to avoid complete growth inhibition. Improvements in biomass and propionic acid productivities were achieved when the dialysis system was used. The performance of the dialysis system was improved when a new pH control strategy evaluated in this study was used.     

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.     

Improving fermentation performance of recombinant zymomonas in acetic acid-containing media

In the production of ethanol from lignocellulosic biomass, the hydrolysis of the acetylated pentosans in hemicellulose during pretreatment produces acetic acid in the prehydrolysate. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process that uses a proprietary metabolically engineered strain ofZymomonas mobilis that can coferment glucose and xylose. Acetic acid toxicity represents a major limitation to bioconversion, and cost-effective means of reducing the inhibitory effects of acetic acid represent an opportunity for significant increased productivity and reduced cost of producing fermentation fuel ethanol from biomass. In this study, the fermentation performance of recombinant Z.mobilis 39676:pZB4L, using a synthetic hardwood prehydrolysate containing 1% (w/v) yeast extract, 0.2% KH₂PO₄, 4% (w/v) xylose, and 0.8% (w/v) glucose, with varying amounts of acetic acid was examine. To minimize the concentration of the inhibitory undissociated form of acetic acid, the pH was controlled at 6.0. The final cell mass concentration decreased linearly with increasing level of acetic acid over the range 0-0.75% (w/v), with a 50% reduction at about 0.5% (w/v) acetic acid. The conversion efficiency was relatively unaffected, decreasing from 98 to 92%. In the absence of acetic acid, batch fermentations were complete at 24 h. In a batch fermentation with 0.75% (w/v) acetic acid, about two-thirds of the xylose was not metabolized after 48 h. In batch fermentations with 0.75% (w/v) acetic acid, increasing the initial glucose concentration did not have an enhancing effect on the rate of xylose fermentation. However, nearly complete xylose fermentation was achieved in 48 h when the bioreactor was fed glucose. In the fed-batch system, the rate of glucose feeding (0.5 g/h) was designed to simulate the rate of cellulolytic digestion that had been observed in a modeled SSCF process with recombinant Zymomonas. In the absence of acetic acid, this rate of glucose feeding did not inhibit xylose utilization. It is concluded that the inhibitory effect of acetic acid on xylose utilization in the SSCF biomass-to-ethanol process will be partially ameliorated because of the simultaneous saccharification of the cellulose.

     

Production of methyltin compounds related to possible conditions in the environment

The methylation of heavy-metal compounds (e.g. mercury, lead, tin) in the environment has great significance owing to the much higher toxicity of their methyl derivatives in comparison with inorganic metal species. In this paper abiological methylation of inorganic tin is described. Ethanol, acetic acid and propionic acid abiologically methylated inorganic tin, and the highest yield of methyltin was observed in the reaction between inorganic tin(II) and ethanol. Furthermore, environmental factors for the methylation, such as pH, temperature, added ethanol, concentration of sodium chloride and photoirradiation, were investigated in this reaction. Methyltin production increased at low pH, and decreased at higher concentrations of sodium chloride. Photoirradiation accelerated the reaction rate, and a shorter wavelength showed a higher rate. Inorganic tin(II) was converted rapidly into monomethyltin, and gradually transformed into dimethyltin and trimethyltin with the course time.     

Production of acetic acid by immobilized whole cells ofClostridium thermoaceticum

Immobilized cells ofClostridium thermoaceticum for acetic acid production has been investigated. Using κ-carrageenan gel as the immobilization-matrix, high cell concentration within the gel could be achieved and thus lead to high volumetric acetic acid productivity. Batch experiments using 3% gel showed that cell concentration up to 65 g (dry cell weight)/L gel could be achieved. These dry weight cell concentrations in the gel through immobilization are typically 10–15 times greater than what can be obtained in free-cell fermentations. The specific growth rate and acetic acid formation rate were similar to those observed for the free cells. Continuous culture experiments using a feed medium containing 20 g/L of glucose were performed where the reactor contained 50% by volume of the carrageenan gel and the pH was controlled at 6.9. Different steady states were acheived at dilution rates ranging from 0.061 to 0.399 h^?1. Cells grew mainly near the surface of the gel and reached maximum concentration within the matrix of approximately 35 g/L. Dilution rates much greater than the maximum specific growth rate were obtained, which resulted in volumetric productivity up to 4.9 g/L-h. This value was significantly greater than that for the conventional continuous culture with free cells. Using a 40 g/L feed glucose concentration, steady states could be achieved between dilution rates of 0.12–0.4 h^?1. The maximum productivity further increased to 6.9 g/L-h at a dilution rate of 0.37 h^?1 and at an acetic acid concentration of 19 g/L. The cell concentration was 60 g (dry weight)/L gel at steady state.     

Culture medium optimization for acetic acid production by a persimmon vinegar-derived bacterium

A new acetic acid-producing microorganism, Acetobacter sp. RKY4, was isolated from Korean traditional persimmon vinegar, and we optimized the culture medium for acetic acid production from ethanol using the newly isolated Acetobacter sp. RKY4. The optimized culture medium for acetic acid production using this microorganism was found to be 40 g/L ethanol, 10 g/L glycerol, 10 g/L corn steep liquor, 0.5 g/L MgSO₄·7H₂O, and 1.0 g/L (NH₄H₂PO₄. Acetobacter sp. RKY4 produced 47.1 g/L of acetic acid after 48 h of fermentation in a 250 mL Erlenmeyer flask containing 50 mL of the optimized medium.     

Immobilization ofLactobacillus bulgaricus in a hollowfiber bioreactor for production of lactic acid from acid whey permeate

Lactobacillus bulgaricus was immobilized in the shell side of an industrial hollow-fiber ultrafiltration module. Acid whey permeate, containing 46 g/L lactose supplemented with 10 g/L yeast extract, was pumped through the tube side at dilution rates of 0.2–2.5/h. At a cell concentration of 100 g/L, productivity was 1.5–5 g lactic acid/L/h.     

Enzyme production, growth, and adaptation of T. reesei strains QM9414, L-27, RL-P37, and rut C-30 to conditioned yellow poplar sawdust hydrolysate

National Renewable Energy Laboratory (NREL) has developed a conditioning process that decreases acetic acid levels in pretreated yellow poplar hydrolysate. Trichoderma reesei is sensitive to acetic acid and this conditioning method has enabled applied cellulase production with hardwoods. T. reesei strains QM9414, L-27, RL-P37, and Rut C-30 were screened for growth on conditioned hydrolysate liquor. Tolerance to hydrolysate was found to be strain-dependent. Strain QM9414 was adapted to grow in 80% (v/v) conditioned hydrolysate (40 g/L of soluble sugars and 1.6 g/L acetic acid from pretreated poplar). However, enzyme production was highest at 20% (v/v) hydrolysateusing strain L-27. Cellulasetiters of 2–3 International Filter Paper Units (IFPU)/mL were achieved using pretreated yellow poplar liquors and solids as the sole carbon sources.     

Preparation of γ-aminobutyric acid using E. coli cells with high activity of glutamate decarboxylase

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter synthesized in the central nervous system from glutamate by glutamate decarboxylase (GAD). It has applications in the production of many drugs. The technology of GABA synthesis by treating L-glutamic acid with the cells of the gene-engineered GAD superproducer strain of Escherichia coli GAD K10 was developed. Cell growing in the presence of 0.02 mM pyridoxal phosphate (PLP) causes the 2- to 2.5-fold increase of total productivity of the cells. The best way to prepare the cells for the reaction was their thermal activation by pretreatment for 1 h at 53°C. The optimal conditions for this reaction were 37°C and pH 4.6. The rate of the enzymatic reaction is the function of acetate concentration with the maximum at 0.5 M acetate. The total amount of GABA synthesized using 1 g of wet cells reached 23–25 g. The final concentration of GABA in the reaction medium was 280–300 g/L. The yield of the product was about 99%.     

Characterization of a Recombinant Flocculent Saccharomyces cerevisiae Strain that Co-ferments Glucose and Xylose: I. Influence of the Ratio of Glucose/Xylose on Ethanol Production

Glucose/xylose mixtures (90 g/L total sugar) were evaluated for their effect on ethanol fermentation by a recombinant flocculent Saccharomyces cerevisiae, MA-R4. Glucose was utilized faster than xylose at any ratio of glucose/xylose, although MA-R4 can simultaneously co-ferment both sugars. A high percentage of glucose can increase cell biomass production and therefore increase the rate of glucose utilization (1.224 g glucose/g biomass/h maximum) and ethanol formation (0.493 g ethanol/g biomass/h maximum). However, the best ratio of glucose/xylose for the highest xylose consumption rate (0.209 g xylose/g biomass/h) was 2:3. Ethanol concentration and yield increased and by-product (xylitol, glycerol, and acetic acid) concentration decreased as the proportion of glucose increased. The maximum ethanol concentration was 41.6 and 21.9 g/L after 72 h of fermentation with 90 g/L glucose and 90 g/L xylose, respectively, while the ethanol yield was 0.454 and 0.335 g/g in 90 g/L glucose and 90 g/L xylose media, respectively. High ethanol yield when a high percentage of glucose is available is likely due to decreased production of by-products, such as glycerol and acetic acid. These results suggest that ethanol selectivity is increased when a higher proportion of glucose is available and reduced when a higher proportion of xylose is available.     

Lactic acid production through cell-recycle repeated-batch bioreactor

The effect of various nitrogen sources on cell growth and lactic acid production was investigated. The most effective nitrogen source was yeast extract; more yeast extract gave higher cell growth and lactic acid productivity. Yeast extract dosage and cell growth were proportional up to a yeast extract concentration of 30 g/L, and lactic acid productivity was linearly correlated up to a yeast extract dosage of 25 g/L. However, increasing the yeast extract content raises the total production cost of lactic acid. Therefore, we attempted to find the optimum yeast extract dosage for a repeated-batch operation with cell recycling. The results show that when using Enterococcus faecalis RKY1 only 26% of the yeast extract dosage for a conventional batch fermentation was sufficient to produce the same amount of lactic acid, whereas the lactic acid concentration in the product stream (92–94 g/L) and lactic acid productivity (6.03–6.20 g/[L·h]) were similar to those of a batch operation. Furthermore, long-term stability was established.