Production of lactic acid from cellobiose and cellotriose by Lactobacillus delbrueckii mutant Uc-3

Lactobacillus delbrueckii mutant Uc-3 utilizes both cellobiose and cellotriose efficiently, converting it into L(+) lactic acid. The enzyme activities of cellobiose and cellotriose utilization were determined for cell extracts, whole cells, and disrupted cells. Aryl-beta-glucosidase activity was detected only for whole cells and disrupted cells, suggesting that these activities are cell bound. The mutant produced 90 g/liter of lactic acid from 100 g/liter of cellobiose with 2.25 g/liter/h productivity.     

Display of α-Amylase on the Surface of Lactobacillus casei Cells by Use of the PgsA Anchor Protein, and Production of Lactic Acid from Starch

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying α-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.     

Improved Succinic Acid Production in the Anaerobic Culture of an Escherichia coli pflB ldhA Double Mutant as a Result of Enhanced Anaplerotic Activities in the Preceding Aerobic Culture

Escherichia coli NZN111 is a pflB ldhA double mutant which loses its ability to ferment glucose anaerobically due to redox imbalance. In this study, two-stage culture of NZN111 was carried out for succinic acid production. It was found that when NZN111 was aerobically cultured on acetate, it regained the ability to ferment glucose with succinic acid as the major product in subsequent anaerobic culture. In two-stage culture carried out in flasks, succinic acid was produced at a level of 11.26 g/liter from 13.4 g/liter of glucose with a succinic acid yield of 1.28 mol/mol glucose and a productivity of 1.13 g/liter·h in the anaerobic stage. Analyses of key enzyme activities revealed that the activities of isocitrate lyase, malate dehydrogenase, malic enzyme, and phosphoenolpyruvate (PEP) carboxykinase were greatly enhanced while those of pyruvate kinase and PEP carboxylase were reduced in the acetate-grown cells. The two-stage culture was also performed in a 5-liter fermentor without separating the acetate-grown NZN111 cells from spent medium. The overall yield and concentration of succinic acid reached 1.13 mol/mol glucose and 28.2 g/liter, respectively, but the productivity of succinic acid in the anaerobic stage dropped to 0.7 g/liter·h due to cell autolysis and reduced anaplerotic activities. The results indicate the great potential to take advantage of cellular regulation mechanisms for improvement of succinic acid production by a metabolically engineered E. coli strain.     

Utilization of Molasses Sugar for Lactic Acid Production by Lactobacillus delbrueckii subsp. delbrueckii Mutant Uc-3 in Batch Fermentation

Efficient lactic acid production from cane sugar molasses by Lactobacillus delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.     

Enzymatic Properties and Intracellular Localization of the Novel Trichoderma reesei β-Glucosidase BGLII (Cel1A)

This paper describes the characterization of an intracellular β-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific β-glucosidase, having no β-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this β-glucosidase.     

Production of Dihomo-γ-Linolenic Acid by a Δ5-Desaturase-Defective Mutant of Mortierella alpina 1S-4

A mutant, which has low Δ5-desaturase activity, of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, was shown to be a novel potent producer of dihomo-γ-linolenic acid (DHGA). On submerged culture under optimal conditions for 6 days at 28°C in a 10-liter fermentor, the mutant produced 3.2 g of DHGA per liter of culture broth (123 mg/g of dry mycelia), which accounted for 23.4% of the total mycelial fatty acids. Mycelial arachidonic acid amounted to only 19 mg/g of dry mycelia (0.5 g/liter of culture broth), which accounted for 3.7% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (11.0%), stearic acid (12.8%), oleic acid (22.7%), linoleic acid (8.9%), γ-linolenic acid (6.5%), and lignoceric acid (7.8%). More than 97 mol% of the DHGA produced was found in the triglyceride fraction irrespective of the growth temperature employed (12 to 28°C).     

Regulation of β-1, 4-Glucosidase Expression by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 expressed β-glucosidase activity (3 to 8 U/ml) constitutively when grown aerobically in complex medium containing either glycerol, succinate, xylose, galactose, or cellobiose as the carbon source. The addition of a high concentration of glucose (>75 g/liter) repressed β-glucosidase expression (<0.3 U/ml); however, this yeast did produce β-glucosidase when the initial glucose concentration was ≤50 g/liter. When grown aerobically in medium containing glucose plus the above-listed carbon sources, diauxic utilization of the carbon source was observed and the expression of β-glucosidase was glucose repressed. Surprisingly, glucose repression did not occur when the cells were grown anaerobically. When grown anaerobically in medium containing 100 g of glucose per liter, C. wickerhamii produced 6 to 9 U of enzyme per ml and did not demonstrate diauxic utilization of glucose-cellobiose mixtures. To our knowledge, this is the first report of apparent derepression of a glucose-repressed enzyme by anaerobiosis.     

Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.     

Incorporation of [15N]Ammonia by the Cellulolytic Ruminal Bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using ¹⁵NH₃. At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH₃-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH₃. More cell nitrogen was formed from NH₃ during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its ¹⁵N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.     

Fermentation of Cellodextrins by Different Yeast Strains

The fermentation of cellodextrins by eight yeast species capable of fermenting cellobiose was monitored. Only two of these species, Torulopsis molischiana and T. wickerhamii, were able to ferment β-glucosides with a degree of polymerization between one and six. These two species showed exocellular β-glucosidase activity. Four other species were able to ferment cellotriose, and the last two species only fermented cellobiose. These latter six species produced a β-glucosidase capable of attacking cellodextrins, but this enzyme was endocellular.     

Non-Sterilized Fermentative Production of Polymer-Grade L-Lactic Acid by a Newly Isolated Thermophilic Strain Bacillus sp. 2–6

Background

The demand for lactic acid has been increasing considerably because of its use as a monomer for the synthesis of polylactic acid (PLA), which is a promising and environment-friendly alternative to plastics derived from petrochemicals. Optically pure l-lactic acid is essential for polymerization of PLA. The high fermentation cost of l-lactic acid is another limitation for PLA polymers to compete with conventional plastics.

Methodology/Principal Findings

A Bacillus sp. strain 2–6 for production of l-lactic acid was isolated at 55°C from soil samples. Its thermophilic characteristic made it a good lactic acid producer because optically pure l-lactic acid could be produced by this strain under open condition without sterilization. In 5-liter batch fermentation of Bacillus sp. 2–6, 118.0 g/liter of l-lactic acid with an optical purity of 99.4% was obtained from 121.3 g/liter of glucose. The yield was 97.3% and the average productivity was 4.37 g/liter/h. The maximum l-lactic acid concentration of 182.0 g/liter was obtained from 30-liter fed-batch fermentation with an average productivity of 3.03 g/liter/h and product optical purity of 99.4%.

Conclusions/Significance

With the newly isolated Bacillus sp. strain 2–6, high concentration of optically pure l-lactic acid could be produced efficiently in open fermentation without sterilization, which would lead to a new cost-effective method for polymer-grade l-lactic acid production from renewable resources.     

The role of lactic acid adsorption by ion exchange chromatography

Background

The polyacrylic resin Amberlite IRA-67 is a promising adsorbent for lactic acid extraction from aqueous solution, but little systematic research has been devoted to the separation efficiency of lactic acid under different operating conditions.

Methodology/Principal Findings

In this paper, we investigated the effects of temperature, resin dose and lactic acid loading concentration on the adsorption of lactic acid by Amberlite IRA-67 in batch kinetic experiments. The obtained kinetic data followed the pseudo-second order model well and both the equilibrium and ultimate adsorption slightly decreased with the increase of the temperature at 293–323K and 42.5 g/liter lactic acid loading concentration. The adsorption was a chemically heterogeneous process with a mean free energy value of 12.18 kJ/mol. According to the Boyd^_plot, the lactic acid uptake process was primarily found to be an intraparticle diffusion at a lower concentration (<50 g/liter) but a film diffusion at a higher concentration (>70 g/liter). The values of effective diffusion coefficient D_i increased with temperature. By using our Equation (21), the negative values of ΔG° and ΔH° revealed that the adsorption process was spontaneous and exothermic. Moreover, the negative value of ΔS° reflected the decrease of solid-liquid interface randomness at the solid-liquid interface when adsorbing lactic acid on IRA-67.

Conclusions/Significance

With the weakly basic resin IRA-67, in situ product removal of lactic acid can be accomplished especially from an open and thermophilic fermentation system without sterilization.     

Physiological Characterization of Pseudomonas putida DOT-T1E Tolerance to p-Hydroxybenzoate

Pseudomonas putida DOT-T1E was isolated as a toluene-tolerant strain. We show that it is also able to grow on high concentrations (up to 17 g/liter [123 mM]) of p-hydroxybenzoate (4HBA). Tolerance to this aromatic carboxylic acid (up to 30 g/liter [217 mM]) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism. The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the cis/trans ratio of unsaturated fatty acids. In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.     

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Rates of phosphorolytic cleavage of β-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by ≥20-fold for both cellobiose and cellopentaose over a 10-fold range of β-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of β-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V_max values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K_m for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

Purification and Partial Characterization of a Glucan Containing Indole-3-acetic Acid

The “bound auxin” of Zea mays, first described by Berger and Avery (Amer. J. Bot. 1944; 31: 199-203) has been purified and partially characterized. It is an indole-3-acetic acid-containing, high molecular weight, lipophilic cellulosicglucan. The indole-3-acetic acid is in ester linkage as evidenced by indoleacetamide formation upon ammonolysis. The glucan is of variable chain length and comprises, in general, 35 to 50 per cent of the dry weight of the compound. The glucosidic residues are β 1 → 4 linked and are hydrolyzed by cellulase. Mild acid hydrolysis produces cellobiose and cellotriose. Other components, as yet unidentified, of the compound are described.     

Kinetics and Modeling of Lactic Acid Production by Lactobacillus plantarum

An unstructured model was developed to describe bacterial growth, substrate utilization, and lactic acid production by Lactobacillus plantarum in cucumber juice. Significant lactic acid production occurred during growth, as well as stationary phases. The percentage of acid produced after growth ceased was a function of the medium composition. Up to 51% of the lactic acid was produced after growth ceased when NaCl was not present in the medium, whereas not more than 18% of the total lactic acid was produced after the growth ceased in presence of NaCl, probably because of an increase in the cell death rate. An equation relating the specific death rate and NaCl concentration was developed. With the kinetic model proposed by R. Luedeking and E. L. Piret (J. Biochem. Microbiol. Technol. Eng. 1:393-412, 1958) for lactic acid production rate, the growth-associated and non-growth-associated coefficients were determined as 51.9 (±4.2) mmol/g of cells and 7.2 (±0.9) mmol/g of cells h^-1 respectively. The model was demonstrated for batch growth of L. plantarum in cucumber juice. Mathematical simulations were used to predict the influence of variations in death rate, proton concentration when growth ceased, and buffer capacity of the juice on the overall fermentation process.     

Efficient production of L-Lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene

We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the l-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2microm plasmid-based vectors and two genome-integrated strains.     

High-Level Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Fed-Batch Culture of Recombinant Escherichia coli

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying alpha-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.