Lactate concentration in breast cancer using advanced magnetic resonance spectroscopy.

BACKGROUND: Precision medicine in breast cancer demands markers sensitive to early treatment response. Aerobic glycolysis (AG) upregulates lactate dehydrogenase A (LDH-A) with elevated lactate production; however, existing approaches for lactate quantification are either invasive or impractical clinically. METHODS: Thirty female patients (age 39-78 years, 15 grade II and 15 grade III) with invasive ductal carcinoma were enrolled. Lactate concentration was quantified from freshly excised whole tumours with double quantum filtered (DQF) magnetic resonance spectroscopy (MRS), and Nottingham Prognostic Index (NPI), LDH-A and proliferative marker Ki-67 were assessed histologically. RESULTS: There was a significantly higher lactate concentration (t = 2.2224, p = 0.0349) in grade III (7.7 ± 2.9 mM) than in grade II (5.5 ± 2.4 mM). Lactate concentration was correlated with NPI (ρ = 0.3618, p = 0.0495), but not with Ki-67 (ρ = 0.3041, p = 0.1023) or tumour size (r = 0.1716, p = 0.3645). Lactate concentration was negatively correlated with LDH-A (ρ = -0.3734, p = 0.0421). CONCLUSION: Our results showed that lactate concentration in whole breast tumour from DQF MRS is sensitive to tumour grades and patient prognosis.

Recent Advances on Purification of Lactic Acid.

With increasing interest in developing biodegradable polymers to replace fossil-based products globally, lactic acid (LA) has been paid extensive attention due to the high environment-compatibility of its downstream products. The mainstream efforts have been put in developing energy-efficient conversion technologies through biological and chemical routes to synthesize LA. However, to our best knowledge, there is a lack of sufficient attention in developing effective separation technologies with high atom economics for purifying LA and derivatives. In this review, the most recent advances in purifying LA using precipitation, reactive extraction, emulsion liquid membrane, reactive distillation, molecular distillation, and membrane techniques will be discussed critically with respect to the fundamentals, flow scheme, energy efficiency, and equipment. The outcome of this article is to offer insights into implementing more atomic and energy-efficient technologies for upgrading LA.

Positive Impact of Pulsed Electric Field on Lactic Acid Removal, Demineralization and Membrane Scaling during Acid Whey Electrodialysis.

The drying of acid whey is hindered by its high mineral and organic acid contents, and their removal is performed industrially through expensive and environmentally impacting serial processes. Previous works demonstrated the ability to remove these elements by electrodialysis alone but with a major concern-membrane scaling. In this study, two conditions of pulsed electric field (PEF) were tested and compared to conventional DC current condition to evaluate the potential of PEF to mitigate membrane scaling and to affect lactic acid and salt removals. The application of a PEF 25 s/25 s pulse/pause combination at an initial under-limiting current density allowed for decreasing the amount of scaling, the final system electrical resistance by 32%, and the relative energy consumption up to 33%. The use of pulsed current also enabled better lactic acid removal than the DC condition by 10% and 16% for PEF 50 s/10 s and 25 s/25 s, respectively. These results would be due to two mechanisms: (1) the mitigation of concentration polarization phenomenon and (2) the rinsing of the membranes during the pause periods. To the best of our knowledge, this was the first time that PEF current conditions were used on acid whey to both demineralize and deacidify it.

Efficient in situ separation and production of L-lactic acid by Bacillus coagulans using weak basic anion-exchange resin.

To get rid of the dependence on lactic acid neutralizer, a simple and economical approach for efficient in situ separation and production of L-lactic acid was established by Bacillus coagulans using weak basic anion-exchange resin. During ten tested resins, the 335 weak basic anion-exchange resins demonstrated the highest adsorption capacity and selectivity for lactic acid recovery. The adsorption study of the 335 resins for lactic acid confirmed that it is an efficient adsorbent under fermentation condition. Langmuir models gave a good fit to the equilibrium data at 50 °C and the maximum adsorption capacity for lactic acid by 335 resins was about 402 mg/g. Adsorption kinetic experiments showed that pseudo-second-order kinetics model gave a good fit to the adsorption rate. When it was used for in situ fermentation, the yield of L-lactic acid by B. coagulans CC17 was close to traditional fermentation and still maintained at about 82% even after reuse by ten times. These results indicated that in situ separation and production of L-lactic acid using the 335 resins were efficient and feasible. This process could greatly reduce the dosage of neutralizing agent and potentially be used in industry.

A Tube-Integrated Painted Biosensor for Glucose and Lactate.

Developing a simple and direct approach for sensitive, specific, and rapid detection of metabolic compounds is of great importance for a variety of biological, medical, and food applications. Tubes are a highly portable and accessible container shape which are widely used for scientific research in cell biology and chemical synthesis, and which are also of great use in domestic health care applications. Here, we show for the first time the development of a tube-based painted amperometric biosensor for the detection of glucose and lactate. The sensor was prepared by printing carbon graphite and silver/silver chloride inks on the interior wall of the tube and then immobilizing glucose oxidase or lactate oxidase on the sensor. The sensor showed a sensitive, rapid, and reliable detection of glucose and lactate. We anticipate that these results could open new avenues for the development of painted biosensors, and toward advanced biosensor applications.

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose.

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapAA1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6g/L, with the yield of 0.48g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6g/L 3-HP at a yield of 0.51g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.

ROS mediated selection for increased NADPH availability in Escherichia coli.

The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli.

Recombinant Lactococcus lactis for efficient conversion of cellodextrins into L-lactic acid.

Lactic acid bacteria (LAB) are among the most interesting organisms for industrial processes with a long history of application as food starters and biocontrol agents, and an underexploited potential for biorefineries converting biomass into high-value compounds. Lactic acid (LA), their main fermentation product, is among the most requested chemicals owing to its broad range of applications. Notably, LA polymers, that is, polylactides, have high potential as biodegradable substitutes of fossil-derived plastics. However, LA production by LAB fermentation is currently too expensive for polylactide to be cost-competitive with traditional plastics. LAB have complex nutritional requirements and cannot ferment inexpensive substrates such as cellulose. Metabolic engineering could help reduce such nutritional requirements and enable LAB to directly ferment low-cost polysaccharides. Here, we engineered a Lactococcus lactis strain which constitutively secretes a ß-glucosidase and an endoglucanase. The recombinant strain can grow on cellooligosaccharides up to at least cellooctaose and efficiently metabolizes them to L-LA in single-step fermentation. This is the first report of a LAB able to directly metabolize cellooligosaccharides longer that cellohexaose and a significant step toward cost-sustainable consolidated bioprocessing of cellulose into optically pure LA.

Isolation and identification of l/d-lactate-conjugated bufadienolides from toad eggs revealing lactate racemization in amphibians.

Three pairs of bufadienolide l/d-lactate epimers (1-6) were isolated from the eggs of the toad Bufo bufo gargarizans. The structures were elucidated by using spectroscopic methods, X-ray diffraction analysis and a modified Mosher's method. Compounds 1-6 represent the first occurrence of lactate-conjugated bufadienolides in nature, and illustrate the existence of an enzyme-controlled epimerization from l- to d-lactate in amphibians. The biosynthetic pathways, in which two key enzymes might be involved (i.e., lactate racemase and acyltransferase), were proposed. In addition, the biological assays revealed that compounds 1-4 are potent cytotoxic agents against human gastric cancer cells BGC-823 and human lung cancer cells A549 with IC50 values in a range of 8.0 to 80.0 nM.

Functional balance between enzymes in malonyl-CoA pathway for 3-hydroxypropionate biosynthesis.

3-Hydroxypropionate (3HP) is an important platform chemical, and four 3HP biosynthetic routes were reported, in which the malonyl-CoA pathway has some expected advantages but presented the lowest 3HP yield. Here, we demonstrated that this low yield was caused by a serious functional imbalance between MCR-C and MCR-N proteins, responsible for the two-step reduction of malonyl-CoA to 3HP. Then we minimized the enzyme activity imbalance by directed evolution of rate-limiting enzyme MCR-C and fine tuning of MCR-N expression level. Combined with culture conditions optimization, our engineering approaches increased the 3HP titer 270-fold, from 0.15 g/L to 40.6 g/L, representing the highest 3HP production via malonyl-CoA pathway so far. This study not only significantly improved the 3HP productivity of recombinant Escherichia coli strain, but also proved the importance of metabolic balance in a multistep biosynthetic pathway, which should be always considered in any metabolic engineering study.

Biosynthesis of platform chemical 3-hydroxypropionic acid (3-HP) directly from CO2 in cyanobacterium Synechocystis sp. PCC 6803.

3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. So far large-scale production of 3-HP has been mainly through petroleum-based chemical processes, whose sustainability and environmental issues have attracted widespread attention. With the ability to fix CO2 directly, cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals. In this study, we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp. PCC 6803, and then optimized the system through the following approaches: i) increasing expression of malonyl-CoA reductase (MCR) gene using different promoters and cultivation conditions; ii) enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-CoA carboxylase and biotinilase; iii) improving NADPH supply by overexpressing the NAD(P) transhydrogenase gene; iv) directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis. Together, the efforts led to a production of 837.18 mg L(-1) (348.8 mg/g dry cell weight) 3-HP directly from CO2 in Synechocystis after 6 days cultivation, demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO2 in cyanobacteria. In addition, the results showed that overexpression of the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) gene from Anabaena sp. PCC 7120 and Synechococcus sp. PCC 7942 led to no increase of 3-HP production, suggesting CO2 fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.

Biotechnological production of enantiomerically pure d-lactic acid.

The fermentation process of l-lactic acid is well known. Little importance was attached to d-lactic acid, but in the past 10 years, d-lactic acid gained significantly in importance. d-Lactic acid is an interesting precursor for manufacturing heat-resistant polylactic acid (PLA) bioplastics which can be widely used, for example as packaging material, coatings, for textiles or in the automotive industry.This review provides a comprehensive overview of the most recent developments, including a spectrum of studied microorganisms and their capabilities for the production of d-lactic acid. Additionally, the technological achievements in biotechnological d-lactic acid production including fermentation techniques like fed batch, simultaneous saccharification, and fermentation and continuous techniques are presented. Attention is also turned to suitable alternative substrates and their applicability in fermentation processes. Furthermore, advantages and disadvantages of product recovery and purification are discussed. Economic aspects of PLA are pointed out, and the present industrial producers of lactic acid are briefly introduced.

An Innovative Optical Sensor for the Online Monitoring and Control of Biomass Concentration in a Membrane Bioreactor System for Lactic Acid Production.

Accurate real-time process control is necessary to increase process efficiency, and optical sensors offer a competitive solution because they provide diverse system information in a noninvasive manner. We used an innovative scattered light sensor for the online monitoring of biomass during lactic acid production in a membrane bioreactor system because biomass determines productivity in this type of process. The upper limit of the measurement range in fermentation broth containing Bacillus coagulans was ~2.2 g·L(-1). The specific cell growth rate (µ) during the exponential phase was calculated using data representing the linear range (cell density ≤ 0.5 g·L(-1)). The results were consistently and reproducibly more accurate than offline measurements of optical density and cell dry weight, because more data were gathered in real-time over a shorter duration. Furthermore, µ(max) was measured under different filtration conditions (transmembrane pressure 0.3-1.2 bar, crossflow velocity 0.5-1.5 m·s(-1)), showing that energy input had no significant impact on cell growth. Cell density was monitored using the sensor during filtration and was maintained at a constant level by feeding with glucose according to the fermentation kinetics. Our novel sensor is therefore suitable for integration into control strategies for continuous fermentation in membrane bioreactor systems.

[The concentration of lactate in amniotic fluid in delivery under physiological course of pregnancy].

The study was carried out to establish admissible range of concentrations of lactate using standard biochemical method in amniotic fluid at first period of delivery under physiologically progressing pregnancy. The sampling included 44 women examined during first period of urgent delivery. The samples of amniotic fluid were taken during the first period of delivery using vaginal amnitomy. The concentration of lactate in samples of amniotic fluid was established using enzymatic amperometric technique. The reference values of concentration of lactate were established in the following admissible limits: 4.4-9.4 mmol per l; ratio lactate/creatinine - 17.7-79.4; ratio lactate/protein - 0.044-0.692. The admissible range of concentration of lactate and ratio lactate/creatinine and lactate/protein in amniotic fluid at first period of delivery under physiologically progressing pregnancy was established.

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans.

We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.

Removing chiral contamination of lactate solutions by selective metabolism of the D-enantiomer.

OBJECTIVE: A bio-based process is appealing for purification of L-lactic acid, the major enantiomer of polylactic acid syrup, generated by thermochemical processes at the end of life of PLA-based plastics, from its chiral impurity, D-lactic acid, before reuse. RESULTS: Polylactic acid (PLA), a renewable alternative to petroleum-derived plastics, contains a mixture of L- and D-lactic acid (LA) isomers with the L-isomer dominating (up to 95 %). A novel bio-based process was developed to produce chirally pure L-LA from syrup produced during recycling of PLA-plastics. This process utilizes an engineered Escherichia coli (strain DC1001) containing novel gene deletions (lld, ykg) that eliminated the oxidative metabolism of L-lactate, leaving the membrane-bound D-lactate dehydrogenases to selectively metabolize the D-isomer. Strain DC1001 removed 8.7 g D-lactate l(-1) from a PLA-syrup containing 135 g total lactic acid l(-1) in 24 h. Average rates of removal of D-lactic acid were 0.25 g D-lactate h(-1) (g cell dry weight)(-1) and 0.36 g D-lactate l(-1) h(-1). CONCLUSION: Bio-based purification of PLA-syrup utilizing E. coli strain DC1001 is an attractive process step during recycling of PLA-plastics. This selective oxidation process can also be used to remove chiral contamination of L-lactate in medical applications.

L (+)-lactic acid production by pellet-form Rhizopus oryzae NRRL 395 on biodiesel crude glycerol.

BACKGROUND: Given its availability and low price, glycerol derived from biodiesel industry has become an ideal feedstock for the production of fuels and chemicals. A solution to reduce the negative environmental problems and the cost of biodiesel is to use crude glycerol as carbon source for microbial growth media in order to produce valuable organic chemicals. In the present paper, crude glycerol was used as carbon substrate for production of L (+)-lactic acid using pelletized fungus R. oryzae NRRL 395 on batch fermentation. More, the experiments were conducted on media supplemented with inorganic nutrients and lucerne green juice. RESULTS: Crude and pure glycerols were first used to produce the highest biomass yield of R. oryzae NRRL 395. An enhanced lactic acid production then followed up using fed-batch fermentation with crude glycerol, inorganic nutrients and lucerne green juice. The optimal crude glycerol concentration for cultivating R. oryzae NRRL 395 was 75 g l(-1), which resulted in a fungal biomass yield of 0.72 g g(-1) in trial without lucerne green juice addition and 0.83 g g(-1) in trial with lucerne green juice. The glycerol consumption rate was 1.04 g l(-1) h(-1) after 48 h in trial with crude glycerol 75 g l(-1) while in trial with crude glycerol 10 g l(-1) the lowest rate of 0.12 g l(-1) h(-1) was registered. The highest L (+)-lactic acid yield (3.72 g g(-1)) was obtained at the crude glycerol concentration of 75 g l(-1) and LGJ 25 g l(-1), and the concentration of lactic acid was approximately 48 g l(-1). CONCLUSIONS: This work introduced sustainable opportunities for L (+)-lactic acid production via R. oryzae NRRL 395 fermentation on biodiesel crude glycerol media. The results showed good fungal growth on crude glycerol at 75 g l(-1) concentration with lucerne green juice supplementation of 25 g l(-1). Lucerne green juice provided a good source of nutrients for crude glycerol fermentation, without needs for supplementation with inorganic nutrients. Crude glycerol and lucerne green juice ratio influence the L (+)-lactic acid production, increasing the lactate productivity with the concentration of crude glycerol.

Utilization of lactic acid bacterial genes in Synechocystis sp. PCC 6803 in the production of lactic acid.

Metabolic pathway engineering of cyanobacteria for the production of industrially important chemicals from atmospheric CO2 has generated interest recently. Here, we engineered Synechocystis sp. PCC 6803 to produce lactic acid using a lactate dehydrogenase (ldh) gene from various lactic acid-producing bacteria, Lactococcus lactis (ldhB and ldhX), Lactobacillus plantarum (ldhL and ldh), and Lactobacillus rhamnosus (ldhL). The lactic acid was secreted outside the cell using a transporter (lldp) gene from L. plantarum. Expression of each ldh in Synechocystis sp. PCC6803 was ascertained by reverse-transcriptase polymerase chain reaction. Five transformants led to the production of L-lactic acid. Co-expression of lldp with ldhB from L. plantarum or ldhL from L. rhamnosus led to the secretion of lactic acid into the medium at concentration of 0.17 ± 0.02 or 0.14 ± 0.02 mM after 18 d of cultivation.

Identification of antifungal compounds produced by Lactobacillus casei AST18.

Lactobacillus casei AST18 was screened as an antifungal lactic acid bacteria which we have reported before. In this research, the antifungal properties of cell-free culture filtrate (CCF) from L. casei AST18 were detected, and the antifungal compounds of CCF were prepared by ultrafiltration, and semi-preparative HPLC, and then determined by GC-MS. CCF was sensitive to pH and heat treatment but it was not affected by the treatment of trypsin and pepsin. Through the treatment of ultrafiltration and semi-preparative HPLC there were two parts of CCF which showed antifungal activities: part 1 and part 4. Lactic acid was identified as the main antifungal compound in part 1. In part 4, three small molecular substances were detected with GC-MS. The three potential antifungal substances were cyclo-(Leu-Pro), 2,6-diphenyl-piperidine, and 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a;1',2'-d]pyrazine. The antifungal activity of L. casei AST18 was a synergistic effect of lactic acid and cyclopeptides.

Sensitive determination of D-lactic acid and L-lactic acid in urine by high-performance liquid chromatography-tandem mass spectrometry.

D-lactic acid in urine originates mainly from bacterial production in the intestinal tract. Increased D-lactate excretion as observed in patients affected by short bowel syndrome or necrotizing enterocolitis reflects D-lactic overproduction. Therefore, there is a need for a reliable and sensitive method able to detect D-lactic acid even at subclinical elevation levels. A new and highly sensitive method for the simultaneous determination of L- and D-lactic acid by a two-step procedure has been developed. This method is based on the concentration of lactic acid enantiomers from urine by supported liquid extraction followed by high-performance liquid chromatography-tandem mass spectrometry. The separation was achieved by the use of an Astec Chirobiotic™ R chiral column under isocratic conditions. The calibration curves were linear over the ranges of 2-400 and 0.5-100 µmol/L respectively for L- and D-lactic acid. The limit of detection of D-lactic acid was 0.125 µmol/L and its limit of quantification was 0.5 µmol/L. The overall accuracy and precision were well within 10% of the nominal values. The developed method is suitable for production of reference values in children and could be applied for accurate routine analysis.