L-乳酸调控乳酸产生菌产物光学纯度的分析

发酵初期在米根霉菌发酵培养基中添加L-乳酸可以调控发酵产物乳酸的光学纯度。随着L-乳酸添加量的增加,所产L-乳酸的光学纯度随之增加,当L-乳酸的添加量≥1.5g/L时,D-乳酸不再产生。同时,L-乳酸的产量、生物量、糖转化率也随之降低。该调控方法对乳酸菌调控产L-乳酸光学纯度影响不大,对大肠杆菌发酵调控产D-乳酸光学纯度没有效果。     

Production of L(+)-lactic acid with Rhizopus oryzae immobilized in polyurethane foam cubes

Summary Polyurethane foam cubes were employed as carriers to immobilize Rhizopus oryzae for L(+)-lactic acid production. The immobilizing capacity reached 450 g-fresh cell/l-cube. The production rate of L(+)-lactic acid could be threefold increased by using the immobilized R. oryzae. The immobilized cells could be steadily used in repetitive fermentations for more than 10 batches.     

Characteristics of immobilized Rhizopus oryzae in polyurethane foam cubes

Summary Rhizopus oryzae was immobilized in polyurethane foam cubes. The effects of the cube size on cell immobilization, cell growth and L(+)-lactic acid production were studied. By the natural attachment method, R. oryzae could be easily immobilized in the polyurethane foam cubes larger than 2.5 × 5 × 5 mm³. The use of small cubes for R. oryzae immobilization was very effective to increase the productivity of L(+)-lactic acid by the immobilized cells. Although it was difficult for smaller cubes to be completely full of the mycelia, increasing the inoculum size in immobilizations was effective to increase the immobilization ratio (a ratio of the number of the cubes containing cells to the total number of cubes).     

Direct fermentation of starch to L-(+)-lactic acid using Lactobacillus amylophilus

Summary Fermentation of L-(+)-lactic acid from soluble starch by Lactobacillus amylophilus was studied. The bacterium produced about 30 g of L-(+)-lactic acid from 50 g of soluble starch when the pH of the culture was ranging from pH 5 to pH 6.8 at 28°C. 53.4 g of L-(+)-lactic acid was produced when 100 g of starch was added in the medium. The fermentation procedures will reduce the cost of complete hydrolysis of starch to glucose prior to fermentation.     

Mutation-Screening in <Emphasis Type="SmallCaps">l</Emphasis>-(+)-Lactic Acid Producing Strains by Ion Implantation

In this paper, in order to obtain some industrial strains with high yield of l-(+)-lactic acid, the wild type strain Lactobacillus casei CICC6028 was mutated by nitrogen ions implantation. By study, it was found that the high positive mutation rate was obtained when the output power was 10 keV and the dose of N⁺ implantation was 50 × 2.6 × 10¹³ ions/cm². In addition, the initial screening methods were also studied, and it was found that the transparent halos method was unavailable, for some high yield strains of l-(+)-lactic acid were missed. Then a mutant strain which was named as N-2 was isolated, its optimum fermentation temperature was 40°C and the l-(+)-lactic acid yield was 136 g/l compared to the original strain whose optimum fermentation temperature was 34°C and l-(+)-lactic acid production was 98 g/l. Finally, High Performance Liquid Chromatography method was used to analyze the purity of l-(+)-lactic acid that was produced by the mutant N-2, and the result showed the main production of N-2 was l-(+)-lactic acid.     

Production of D(-)-lactic acid from cellulose by simultaneous saccharification and fermentation using Lactobacillus coryniformis subsp. torquens

D(–)-Lactic acid was produced from cellulose by simultaneous saccharification and fermentation (SSF) in media containing cellulolytic enzymes and Lactobacillus coryniformis subsp. torquens ATCC 25600 at 39 °C and pH 5.4, yielding 0.89 g D(–)-lactic acid g^–1 cellulose at a mean volumetric productivity of 0.5 g l^–1 h^–1. No L(+)-lactic acid was found in the medium.     

Fermentation of sugar cane bagasse hemicellulose hydrolysate to l(+)-lactic acid by a thermotolerant acidophilic Bacillus sp.**

Sugar cane bagasse hemicellulose, hydrolyzed by dilute H2SO4, supplemented with mineral salts and 0.5% corn steep liquor, was fermented to L(+)-lactic acid using a newly isolated strain of Bacillus sp. In batch fermentations at 50 degrees C and pH 5, over 5.5% (w/v) L(+)-lactic acid was produced (89% theoretical yield; 0.9 g lactate per g sugar) with an optical purity of 99.5%.     

Optimization of L(+)-lactic acid production employing statistical experimental design

Summary An orthogonal 2³-factorial experimental design was used to optimize L(+)-lactic acid production byLactobacillus casei. With a 22 % (v/v) inoculum the optimum concentration of yeast extract for maximum lactic acid concentration and yield was about 2 % (w/v) and that of the initial glucose 9 to 11 %.     

Direct production of L+-lactic acid from starch and food wastes using Lactobacillus manihotivorans LMG18011

This study describes several essential factors for direct and effective lactic acid production from food wastes by Lactobacillus manihotivorans LMG18011, and optimum conditions for simultaneous saccharification and fermentation using soluble starch and food wastes as substrates. The productivity was found to be affected by three factors: (1) initial pH, which influenced amylase production for saccharification of starch, (2) culture pH control which influenced selective production of L(+)-lactic acid, and (3) manganese concentration in medium which improved in production rate and yield of lactic acid. The optimum initial pH was 5.0-5.5, and the fermentation pH for the direct and effective fermentation from starchy substrate was 5.0 based on the yield of L(+)-lactic acid. Under these conditions, 19.5 g L(+)-lactic acid was produced from 200 g food wastes by L. manihotivorans LMG18011. Furthermore, the addition of manganese stimulated the direct fermentation significantly, and enabled complete bioconversion within 100 h.     

Application of L-(+)-Lactate electrode for clinical analysis and monitoring of tissue culture medium

L-(+)-Lactate oxidase (EC 1.1.3.2) was immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure which has selective permeability to hydrogen peroxide. The lactate electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized enzyme membrane. Properties of the enzyme membrane and characteristics of the lactate electrode were clarified for the determination of L-(+)-lactic acid. The lactate electrode responded linearly to L-(+)-lactic acid over the final concentration 0-0.25 mmol/L within 30 s. When the enzyme electrode was applied to the determination of L-(+)-lactic acid in control serum, within-day precision (CV), analytical recovery, and correlation coefficient between the electrode method and the colorimetric method were 1.4% with a mean value of 4.54 mmol/L, 98.0%, and 0.986, respectively. The lactate electrode was sufficiently stable to perform 1040 assays over 13 days operation for the determination of L-(+)-lactic acid. The dried immobilized enzyme membrane retained 84% of its initial activity after storage at 4 degrees C for 12 months. Moreover, the enzyme electrode was applied to the monitoring of culture medium for human melanoma cells. L-(+)-Lactate production and D-glucose consumption were closely related to cell numbers.     

Potential of solid state fermentation for production of L(+)-lactic acid by Rhizopus oryzae

Production of l(+)-lactic acid by Rhizopus oryzae NRRL 395 was studied in solid medium on sugar-cane bagasse impregnated with a nutrient solution containing glucose and CaCO₃. A comparative study was undertaken in submerged and solid-state cultures. The optimal concentrations in glucose were 120 g/l in liquid culture and 180 g/l in solid-state fermentation corresponding to production of l(+)-lactic acid of 93.8 and 137.0 g/l, respectively. The productivity was 1.38 g/l per hour in liquid medium and 1.43 g/l per hour in solid medium. However, the fermentation yield was about 77% whatever the medium. These figures are significant for l(+)-lactic acid production.     

Experimental design to enhance the production of l-(+)-lactic acid from steam-exploded wood hydrolysate using Rhizopus oryzae in a mixed-acid fermentation

The fermentation of hemicellulosic hydrolysate from Pinus taeda chips, using the fungal culture Rhizopus oryzae, was carried out to produce l-(+)-lactic acid and to optimize and enhance the biological conversion of reducing sugar into l-(+)-lactic acid using the experimental design to evaluate the culture conditions. The first factorial design based on surface response with five factors (agitation level, substrate concentration, CaCO₃ concentration, C/N and C/P ratios) at low levels and one medium point was performed to optimize culture conditions. The second study tested two factors (substrate concentration and C/N ratio) at three levels. The statistical analysis of the data obtained from the factorial study showed that a C/N ratio of 35 and substrate concentration of 90 g/litre were the best conditions to produce l-(+)-lactic acid with R. oryzae on P. taeda hydrolysate, but in this case the statistical projection was not correct and the real optimized conditions were C/N ratio of 55 and substrate concentration of 75 g/litre of reducing sugar.     

Production of optically pure d-lactic acid from brown rice using metabolically engineered Lactobacillus plantarum

Simultaneous saccharification and fermentation (SSF) of d-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the ʟ-lactate dehydrogenase gene was disrupted, produced 97.7 g/L d-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8–17 h). As a result, 117.1 g/L d-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and d-lactic acid was stably produced using recycled cells (118.4–129.8 g/L). On average, d-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.

     

Membrane-Integrated Fermentation System for Improving the Optical Purity of D-Lactic Acid Produced during Continuous Fermentation

This report describes the production of highly optically pure D-lactic acid by the continuous fermentation of Sporolactobacillus laevolacticus and S. inulinus, using a membrane-integrated fermentation (MFR) system. The optical purity of D-lactic acid produced by the continuous fermentation system was greater than that produced by batch fermentation; the maximum value for the optical purity of D-lactic acid reached 99.8% enantiomeric excess by continuous fermentation when S. leavolacticus was used. The volumetric productivity of the optically pure D-lactic acid was about 12 g/L/h, this being approximately 11-fold higher than that obtained by batch fermentation. An enzymatic analysis indicated that both S. laevolacticus and S. inulinus could convert L-lactic acid to D-lactic acid by isomerization after the late-log phase. These results provide evidence for an effective bio-process to produce D-lactic acid of greater optical purity than has conventionally been achieved to date.     

Untersuchungen zur Entstehung von Dl-Milchsäure bei Lactobacillen und Charakterisierung einer Milchsäureracemase bei einigen Arten der Untergattung Streptobacterium

Zusammenfassung Eine kritische Überprüfung der von den verschiedenen Arten der Gattung Lactobacillus gebildeten Milchsäure ergab, daß zwar die D(-)-Laktatbildner reines D(-)-Isomer, die L(+)-Laktatbildner aber immer auch einige wenige Prozente des anderen Isomers bilden. Letzteres beruht auf der Anwesenheit einer sehr schwach aktiven NAD-abhängigen D-Laktatdehydrogenase neben der hochaktiven NAD-abhängigen L-Laktatdehydrogenase.Die Bildung von Dl-Laktat beruht entweder auf der Anwesenheit der beiden stereospezifisch verschiedenen Laktatdehydrogenasen oder auf der Bildung vo L(+)-Milchsäure und anschließender Racemisierung. In einigen Fällen ist die biochemische Grundlage der Racematbildung noch unklar. Bei fast allen Dl-Bildnern ist das Isomerenverhältnis von den Wachstumsbedingungen abhängig.Bei Lactobacillus curvatus, L. sake und L. casei ssp. pseudoplantarum wurde die für die Dl-Bildung verantwortliche Milchsäureracemase nachgewiesen und näher charakterisiert. Es handelt sich um ein induzierbares Enzym, dessen Induktor L(+)-Milchsäure ist. Die Induktion kann mit Actinomycin D gehemmt werden. Eine Trennung von Racemase und L-Laktatdehydrogenase gelang durch Zentrifugation im Saccharosedichtegradienten, da das Molekulargewicht der Racemase mit 52–60000 in einigen Fällen niedriger liegt als das der Laktatdehydrogenase.

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Formation of Dl-lactic acid by lactobacilli and characterization of a lactic acid racemase from several streptobacteria

Summary The isomer composition of the lactic acid formed by the various species of lactobacilli was enzymatically determined. It is shown, that the D(-)-lactate formers produce D(-)-lactate exclusively whereas all L(+)-lactate formers always produce a few per cent of the other isomer in addition. The latter is due to the presence of a NAD dependent D-LDH of very low activity. The formation of Dl-lactic acid is either caused by the presence of both stereospecific different LDHs or by the formation of L(+)-lactic acid followed by racemisation. In some species the biochemical basis of the racemate formation is still unknown.The racemase was isolated and characterised from Lactobacillus curvatus, L. sake and L. casei ssp. pseudoplantarum. This enzyme is induced by L(+)-lactic acid. The induction is inhibited by actinomycine D. A separation of lactic acid racemase and of L-LDH was achieved by the application of a sucrose density gradient.

     

Metabolic engineering of Lactobacillus helveticus CNRZ32 for production of pure L-(+)-lactic acid

Expression of D-(-)-lactate dehydrogenase (D-LDH) and L-(+)-LDH genes (ldhD and ldhL, respectively) and production of D-(-)- and L-(+)-lactic acid were studied in Lactobacillus helveticus CNRZ32. In order to develop a host for production of pure L-(+)-isomer of lactic acid, two ldhD-negative L. helveticus CNRZ32 strains were constructed using gene replacement. One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene of ldhD with an additional copy of the structural gene (ldhL) of L-LDH of the same species. The resulting strains were designated GRL86 and GRL89, respectively. In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter. The two D-LDH-negative strains produced only L-(+)-lactic acid in an amount equal to the total lactate produced by the wild type. The maximum L-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain. Furthermore, process variables for L-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology. The temperature and pH optima were 41 degrees C and pH 5.9. At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.     

Metabolic engineering of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> for the production of platform chemicals

Rhizopus oryzae is a filamentous fungus belonging to the Zygomycetes. It is among others known for its ability to produce the sustainable platform chemicals l-(+)-lactic acid, fumaric acid, and ethanol. During glycolysis, all fermentable carbon sources are metabolized to pyruvate and subsequently distributed over the pathways leading to the formation of these products. These platform chemicals are produced in high yields on a wide range of carbon sources. The yields are in excess of 85 % of the theoretical yield for l-(+)-lactic acid and ethanol and over 65 % for fumaric acid. The study and optimization of the metabolic pathways involved in the production of these compounds requires well-developed metabolic engineering tools and knowledge of the genetic makeup of this organism. This review focuses on the current metabolic engineering techniques available for R. oryzae and their application on the metabolic pathways of the main fermentation products.     

Effect of oleuropein and sodium chloride on viability and metabolism of Lactobacillus plantarum

 The effect of the addition of oleuropein (OLP) and NaCl on the growth and the DL-lactic acid production of Lactobacillus plantarum DSM 10492 has been investigated by using an unconventional medium. The growth of L. plantarum was not inhibited by the addition of increasing amounts of untreated OLP in the presence or absence of glucose. However, bacterial cells grew in quantity slightly with OLP alone. The increased addition of NaCl was associated with a delay in growth. Moreover, there was no growth with 8% NaCl. The addition of both NaCl and OLP resulted in growth inhibition, and the survival of cells decreased strongly. The main fermentation product was DL-lactic acid, but acetic acid was also detected after a prolonged incubation. L. plantarum produced DL-lactic acid in the presence of OLP alone but its formation decreased with increasing levels of OLP. On the other hand, heat-treated OLP had a bactericidal effect. Received: 16 October 1995/Received last revision: 5 February 1996/Accepted: 12 February 1996     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Enhancement of pilot scale production of l(+)-lactic acid by fermentation coupled with separation using membrane bioreactor

Traditional batch fermentation leads to a higher energy consumption and lower production capability because of longer culture time. In this work, a pilot scale bioreactor composed of a 3000 L fermentor and external ceramic microfiltration equipment was used to perform cell-recycle fermentation. Repeat feeding medium was also used to relieve the substrate inhibition. In such pilot system, the maximum yield and productivity of l(+)-lactic acid production reached 157.22 ± 3.42 g/L and 8.77 ± 0.15 g/L/h which were 4.23% and 315.64% higher than those of batch fermentation, respectively, when equal amount of sugar was consumed. The cost of l(+)-lactic acid production was successfully reduced about two-thirds by the increase of yield and productivity. 12 rounds of cell-recycle fermentations were successfully achieved in the pilot system. The membrane filtration productivity reached to 61.27 ± 2.74 L/m²/h which increased 172.80%, while the cell damaging rate dropped to 3.88 ± 0.18% which decreased 85.77%, compared with those of the ultrafiltration. Furthermore, the ceramic microfiltration membrane showed advantages in tolerance for the temperature, pressure and acid, compared with the organic ultrafiltration membrane. The experimental results indicated that the method could give a reference for low cost production of l(+)-lactic acid in an industrial scale.