Homolactic Acid Research Articles

Changes in chemical properties and microbial communities' composition of a forest litter-based biofertilizer produced through aerated solid-state culture under different oxygen conditions.

Fermented forest litter (FFL) is a bioproduct used as biofertilizer for several decades in Eastern Asia and Latin America. It is locally handcrafted by farmers in anaerobic conditions by fermenting forest litter added with agricultural by-products such as whey, cereal bran, and molasses. The aim of this study was to characterize the FFL process and product through gas and liquid chromatography analyses. It also provides some highlights on the influence of O2 on this solid-state culture. Under anoxic condition, a maximum CO2 production rate (CDPR) of 0.41mL/h∙g dry matter (dm) was reached after 8days. The main volatile organic compounds (VOCs) were ethanol and ethyl acetate, with a production rate profile similar to CDPR. After 21days of culture, no residual sucrose nor lactose was detected. Lactic and acetic acids reached 58.8mg/g dm and 10.2mg/g dm, respectively, ensuring the acidification of the matrix to a final pH of 4.72. A metabarcoding analysis revealed that heterolactic acid bacteria (Lentilactobacillus, Leuconostoc), homolactic acid bacteria (Lactococcus), and yeasts (Saccharomyces, Clavispora) were predominant. Predicted genes in the microbiome confirmed the potential link between detected bacteria and acids and VOCs produced. When O2 was fed to the cultures, final pH reached values up to 8.5. No significant amounts of lactic nor acetic acid were found. In addition, a strong shift in microbial communities was observed, with a predominance of Proteobacteria and molds, among which are potential pathogens like Fusarium species. This suggests that particular care must be brought to maintain anoxic conditions throughout the process.

Technological Improvements on FML in the Chianti Classico Wine Production: Co-Inoculation or Sequential Inoculation?

Winemaking variables and techniques are known to affect the composition of wines. To obtain a rapid and safe fermentation course, with a reduced risk of proliferation of unwanted microbial species, frequent recourse is made to the use of selected microorganisms, which can greatly simplify the complex management of the fermentation process. In particular, selected strains of lactic acid bacteria are used, which are much more sensitive than yeasts to the operating conditions of the medium. In this regard, the overall aim of this research was to verify whether the early inoculation of homolactic acid bacteria for hexoses (Lactobacillus plantarum) carried out after 24 h, compared with that of saccharomycetes operating alcoholic fermentation, could be advantageous compared with a traditional innoculation with a different heterolactic bacterial strain for hexoses (Oenococcus oeni) operated at the end of alcoholic fermentation. The grape variety chosen was Sangiovese, the protagonist of Tuscan oenology. The evaluation focused on different aspects such as the management of winery operations, and the quality and longevity of the product; was carried out in all phases of winemaking; and analysed both from a chemical and sensory point of view.

Effects of cellulolytic lactic acid bacteria on the lignocellulose degradation, sugar profile and lactic acid fermentation of high-moisture alfalfa ensiled in low-temperature seasons

The objective of this study was to investigate the effects of cellulolytic lactic acid bacteria on the lignocellulose degradation, sugar profile and lactic acid fermentation of high-moisture alfalfa during anaerobic bioprocessing in low-temperature seasons. Last cut and low dry matter (DM) alfalfa was ensiled with five additives, commercial cellulase (EN), a combination of Lactobacillus plantarum and commercial cellulase (LPEN), a combination of engineered Lactococcus lactis strains (HT2) containing cellulase gene, Enterococcus faecium Y83 (EF83), or Enterococcus faecalis JF85 (EF85), and without additives, as the control, with four replicates each. After anaerobic preservation in a silo from late fall through winter (3 to 20 °C) for 140 days, the ensiled alfalfa was sampled and analysed. Compared with the control, EF85 increased the relative contents of neutral detergent fibre (NDF), acid detergent fibre (ADF), and cellulose and decreased contents of water-soluble carbohydrates (WSCs) and lactic acid, and the conversion of WSCs into lactic acid. Consequently, lactic acid fermentation was decreased in EF85. Compared with the control, supplementation with EN, EF83, HT2, and LPEN decreased contents of NDF, ADF, cellulose, and hemicellulose and increased the contents of WSCs, disaccharide, xylose, and arabinose; interestingly, compared with EN, supplementation with EF83, HT2 and LPEN increased the lactic acid content and the ratio of lactic acid to acetic acid as indicated by strong homo-lactic acid fermentation. Furthermore, HT2 increased the lactic acid concentration to a maximum of 72.2 g/kg DM and had a higher conversion of WSCs into lactic acid than EF83 (36.3%) and LPEN (17.9%), indicating that HT2 had a greater ability to ferment lignocellulose of alfalfa into lactic acid. Therefore, HT2 could enhance the lactic acid production of alfalfa during ensiling in low-temperature seasons.

Effect of pre-fermentation types on the potential of methane production and energy recovery from food waste

Two-phase anaerobic digestion (TPAD) is a commonly used method for recovering energy from food waste, even though the relationship between fermentation type and methane production has not to be thoroughly investigated. In this study, homolactic acid fermentation (HOLA), heterolactic acid fermentation (HELA), butyric acid fermentation (BUA), and mixed acid fermentation (MA) were used in the first phase, and the corresponding methane production levels were compared. HELA and MA resulted in the maximum methane yields of 290 and 287 ml per gram chemical oxygen demand (COD), respectively, but they were not significantly higher than the yield of 279 ml/g COD from single-phase anaerobic digestion (SPAD). During methanogenesis, BUA led to the fastest hydrolysis and methane production rates, followed by MA and HELA. In spite of the similar potential for methane production and energy recovery, TPAD using either BUA, MA, or HELA as the fermentation phase exhibited at least 50% greater methane production efficiency than SPAD. Overall, HELA and MA were found to be the best choices in terms of treatment efficiency and energy recovery.

Correction for Iwasaki et al., "Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073".

Correction for Iwasaki et al., "Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073".

Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073.

For the efficient production of target metabolites from carbohydrates, syngas, or H2-CO2 by genetically engineered Moorella thermoacetica, the control of acetate production (a main metabolite of M. thermoacetica) is desired. Although propanediol utilization protein (PduL) was predicted to be a phosphotransacetylase (PTA) involved in acetate production in M. thermoacetica, this has not been confirmed. Our findings described herein directly demonstrate that two putative PduL proteins, encoded by Moth_0864 (pduL1) and Moth_1181 (pduL2), are involved in acetate formation as PTAs. To disrupt these genes, we replaced each gene with a lactate dehydrogenase gene from Thermoanaerobacter pseudethanolicus ATCC 33223 (T-ldh). The acetate production from fructose as the sole carbon source by the pduL1 deletion mutant was not deficient, whereas the disruption of pduL2 significantly decreased the acetate yield to approximately one-third that of the wild-type strain. The double-deletion (both pduL genes) mutant did not produce acetate but produced only lactate as the end product from fructose. These results suggest that both pduL genes are associated with acetate formation via acetyl-coenzyme A (acetyl-CoA) and that their disruption enables a shift in the homoacetic pathway to the genetically synthesized homolactic pathway via pyruvate.IMPORTANCE This is the first report, to our knowledge, on the experimental identification of PTA genes in M. thermoacetica and the shift of the native homoacetic pathway to the genetically synthesized homolactic pathway by their disruption on a sugar platform.

Fed-batch fermentation for enhanced lactic acid production from glucose/xylose mixture without carbon catabolite repression

There has been tremendous growth in the production of optically pure l-lactic acid from lignocellulose-derived sugars. In this study, Enterococcus mundtii QU 25 was used to ferment a glucose/xylose mixture to l-lactic acid. Maintenance of the xylose concentration at greater than 10g/L achieved homo-lactic acid fermentation and reduced the formation of byproducts. Furthermore, carbon catabolite repression (CCR) was avoided by maintaining the glucose concentration below 25g/L; therefore, initial concentrations of 25g/L glucose and 50g/L xylose were selected. Supplementation with 5g/L yeast extract enhanced the maximum xylose consumption rate and consequently increased lactic acid production and productivity. Finally, a 129g/L lactic acid without byproducts was obtained with a maximum lactic acid productivity of 5.60g/(L·h) in fed-batch fermentation with feeding a glucose/xylose mixture using ammonium hydroxide as the neutralizing agent. These results indicate a potential for lactic acid production from glucose and xylose as the main components of lignocellulosic biomasses.

Kinetic Investigation In Lactic Acid Production By L. Acidophilus

Lactic acid is a natural organic acid, which is used in chemical, textile, pharmaceuticals, and food industries. Commercial lactic acid is being produced by fermentation of carbohydrate by using lactic acid bacteria. The purpose of this study is to calculate the following kinetic parameters: the maximum specific growth rate , the Monod constant , and the product yield coefficient in the process of production lactic acid from glucose by Lactobacillus Acidophilus, which is a homolactic acid bacteria.Several batch fermentations at glucose concentration range from 9.3 g/L to 151.2 g/L were performed at optimum condition at 37 oC and pH = 6.5. When increasing glucose concentration, value of maximum specific growth rate reduced, thus non-competitive inhibition exists. The maximum specific growth rate was 0.289 hr-1, substrate inhibition constant was 133 ± 3.2 g/L and KS = 0.8 g/L. Yield of product was decreasing from 0.81 to 0.63 when increasing initial concentration. For scale-up process, the initial glucose concentration should not excess 90g/L.

Characterization of Three Lactic Acid Bacteria and Their Isogenic ldh Deletion Mutants Shows Optimization for Y ATP (Cell Mass Produced per Mole of ATP) at Their Physiological pHs

Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y(ATP) (cell mass produced per mole of ATP). The pH that showed the highest Y(ATP) corresponded to the pH of the natural habitat of the organisms.

Shewanella oneidensis in a lactate-fed pure-culture and a glucose-fed co-culture with Lactococcus lactis with an electrode as electron acceptor

Bioelectrochemical systems (BESs) employing mixed microbial communities as biocatalysts are gaining importance as potential renewable energy, bioremediation, or biosensing devices. While we are beginning to understand how individual microbial species interact with an electrode as electron donor, little is known about the interactions between different microbial species in a community: sugar fermenting bacteria can interact with current producing microbes in a fashion that is either neutral, positively enhancing, or even negatively affecting. Here, we compare the bioelectrochemical performance of Shewanella oneidensis in a pure-culture and in a co-culture with the homolactic acid fermenter Lactococcus lactis at conditions that are pertinent to conventional BES operation. While S. oneidensis alone can only use lactate as electron donor for current production, the co-culture is able to convert glucose into current with a comparable coulombic efficiency of ∼17%. With (electro)-chemical analysis and transcription profiling, we found that the BES performance and S. oneidensis physiology were not significantly different whether grown as a pure- or co-culture. Thus, the microbes worked together in a purely substrate based (neutral) relationship. These co-culture experiments represent an important step in understanding microbial interactions in BES communities with the goal to design complex microbial communities, which specifically convert target substrates into electricity.

Effects of combining Lactobacillus buchneri 40788 with various lactic acid bacteria on the fermentation and aerobic stability of corn silage

The objective was to compare the effectiveness of combining Lactobacillus buchneri 40788 with three lactic acid bacteria on the fermentation, aerobic stability and nutritive value of corn silage. Freshly chopped whole plant corn was untreated or treated with L. buchneri 40788 paired with Lactobacillus plantarum, Pediococcus acidilactici, or Pediococcus pentosaceus. L. buchneri was added to achieve 4 × 10 5 CFU/g of fresh forage and the homolactic acid bacteria were added to achieve 1 × 10 5 CFU/g. Forages were ensiled in laboratory silos for 215 days before opening. Overall, inoculation increased the concentrations of acetic acid and 1,2-propanediol, but it reduced the concentrations of lactic acid, water soluble carbohydrates and ethanol. Effects on acetic acid and 1,2-propanediol were biggest for L. buchneri 40788 paired with P. pentosaceus > P. acidilactici > L. plantarum. Combining L. buchneri 40788 with L. plantarum or P. acidilactici increased and tended to increase in vitro aNDF digestibility in silage when compared to untreated silage, respectively. Untreated silage had a relatively large proportion of visibly spoiled silage at the surface of the silos, which resulted in poorer recovery of feedable dry matter when compared to silage treated with the combinations of L. buchneri 40788 with L. plantarum or with P. acidilactici. All inoculated silages had fewer numbers of yeasts after ensiling which improved their aerobic stability relative to untreated silage. Addition of L. buchneri 40788 to corn silage improves its aerobic stability and combining it with the P. acidilactici and L. plantarum strains used in this study results in other beneficial effects on silage quality.

Inhibition kinetics of catabolic dehydrogenases by elevated moieties of ATP and ADP – implication for a new regulation mechanism in Lactococcus lactis

ATP and ADP inhibit, in varying degrees, several dehydrogenases of the central carbon metabolism of Lactococcus lactis ATCC 19435 in vitro, i.e. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH). Here we demonstrate mixed inhibition for GAPDH and competitive inhibition for LDH and ADH by adenine nucleotides in single inhibition studies. The nonlinear negative co-operativity was best modelled with Hill-type kinetics, showing greater flexibility than the usual parabolic inhibition equation. Because these natural inhibitors are present simultaneously in the cytoplasm, multiple inhibition kinetics was determined for each dehydrogenase. For ADH and LDH, the inhibitor combinations ATP plus NAD and ADP plus NAD are indifferent to each other. Model discrimination suggested that the weak allosteric inhibition of GAPDH had no relevance when multiple inhibitors are present. Interestingly, with ADH and GAPDH the combination of ATP and ADP exhibits lower dissociation constants than with either inhibitor alone. Moreover, the concerted inhibition of ADH and GAPDH, but not of LDH, shows synergy between the two nucleotides. Similar kinetics, but without synergies, were found for horse liver and yeast ADHs, indicating that dehydrogenases can be modulated by these nucleotides in a nonlinear manner in many organisms. The action of an elevated pool of ATP and ADP may effectively inactivate lactococcal ADH, but not GAPDH and LDH, providing leverage for the observed metabolic shift to homolactic acid formation in lactococcal resting cells on maltose. Therefore, we interpret these results as a regulation mechanism contributing to readjusting the flux of ATP production in L. lactis.

Ethanol and lactic acid production using sap squeezed from old oil palm trunks felled for replanting

Old oil palm trunks that had been felled for replanting were found to contain large quantities of high glucose content sap. Notably, the sap in the inner part of the trunk accounted for more than 80% of the whole trunk weight. The glucose concentration of the sap from the inner part was 85.2g/L and decreased towards the outer part. Other sugars found in relatively low concentrations were sucrose, fructose, galactose, xylose, and rhamnose. In addition, oil palm sap was found to be rich in various kinds of amino acids, organic acids, minerals and vitamins. Based on these findings, we fermented the sap to produce ethanol using the sake brewing yeast strain, Saccharomyces cerevisiae Kyokai no.7. Ethanol was produced from the sap without the addition of nutrients, at a comparable rate and yield to the reference fermentation on YPD medium with glucose as a carbon source. Likewise, we produced lactic acid, a promising material for bio-plastics, poly-lactate, from the sap using the homolactic acid bacterium Lactobacillus lactis ATCC19435. We confirmed that sugars contained in the sap were readily converted to lactic acid with almost the same efficiency as the reference fermentation on MSR medium with glucose as a substrate. These results indicate that oil palm trunks felled for replanting are a significant resource for the production of fuel ethanol and lactic acid in palm oil-producing countries such as Malaysia and Indonesia.

Effects of inoculation of high dry matter alfalfa silage on ensiling characteristics, ruminal nutrient degradability and dairy cow performance

AbstractThe objective of this study was to determine the effects of a homolactic acid inoculant on ensiling characteristics and nutritive value of high dry matter (DM) alfalfa. The ensiling characteristics were determined by ensiling inoculated and untreated alfalfa haylage in mini‐silos for 0, 2, 4, 8, 16 and 45 days. Two lactating cows fitted with ruminal fistulas were used to determine ruminal degradabilities of nutrient in inoculated and untreated alfalfa silage (45‐day silage). Effects of feeding inoculated and untreated alfalfa silage on animal performance were determined using 28 lactating dairy cows fed total mixed diets (40% forage and 60% concentrate) with the forage portion consisting of inoculated or untreated alfalfa silage. The pH of the inoculated silage declined from 5.9 to 4.5 within 2 days of ensiling while the pH of the untreated silage did not drop below 5 until day 16 post‐ensiling. At all ensiling times, inoculated alfalfa silage had lower pH than untreated alfalfa silage. The concentration of lactic acid was higher while that of water‐soluble carbohydrates was lower for inoculated than untreated alfalfa silage at all ensiling times. Inoculation increased proteolysis as indicated by a reduction in true protein and neutral detergent insoluble protein and an increase in non‐protein nitrogen. Ruminal degradability of DM, crude protein and neutral detergent fiber of alfalfa silage were not affected by inoculation. The average effective degradability of dry matter, crude protein and neutral detergent fiber was 66, 65 and 62%, respectively. Dairy cows fed inoculated alfalfa silage had DM intake (average 22 kg day−1) and milk yield (average 42 kg day−1) similar to cows fed untreated alfalfa silage. It was concluded that the inoculant used in this study improved the ensiling characteristics of alfalfa silage with no significant effects on dairy cow performance. Copyright © 2004 Society of Chemical Industry

Effect of Lactobacillus buchneri 40788 on the fermentation, aerobic stability and nutritive value of maize silage

The effect of adding Lactobacillus buchneri 40788, a heterofermentative lactobacilli, to whole‐plant maize and its effect on fermentation and aerobic stability of the resulting silage were evaluated. Whole‐plant maize (380 g DM kg–1 fresh weight) was ensiled in laboratory silos after the following treatments: untreated, L. buchneri 40788 at 1 × 105, 2·5 × 105, 5 × 105, and 1 × 106 colony forming units g–1 of fresh forage or, a commercial inoculant containing homolactic acid bacteria, a single species of propionibacteria and enzymes. Addition of L. buchneri 40788 resulted in a decrease in lactic acid concentration but increased the concentration of acetic acid in silage and markedly decreased the numbers of yeasts present in the silage. All levels of L. buchneri 40788, increased the aerobic stability of silages but the effect was greatest for the two highest levels of inoculation. Treatment with the commercial inoculant had no effect on the fermentation or aerobic stability of silage. On‐farm treatment of maize silage with L. buchneri 40788, stored in a bag silo, resulted in changes in fermentation that were similar to those observed in laboratory silos. Treatment decreased the numbers of yeast in silage and increased aerobic stability (+25 h) of the silage when exposed to air. Treatment had no effect on the dry‐matter intake of silage fed to sheep. These results showed that treating silage with L. buchneri 40788 can increase the aerobic stability of maize silage in laboratory and farm silos.

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio.

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.

SPOROLACTOBACILLUS NOV. SUBGEN.

A motile, spore-forming and rod-shaped bacterium was isolated from a chiken feed. The strain is Gram-positive, catalase-negative and is a typical homo-lactic acid fermenter producing leavo-rotatory lactic acid. Taxonomic position of this unusual strain was discussed and it was suggested that the organism represents an intermediate form between Lactobacillus and Clostridium, for which erection of a new subgenus Sporolactobacillus (in Lactobacillaceae) was proposed. The species was named Sporolactobacillus inulinus nov. sp. taking into account its powerful inulin fermenting activity.

QUANTITATIVE STUDIES ON GLYCOLYTIC ENZYMES IN LACTOBACILLUS PLANTARUM 11

Glucose-6-phosphate dehydrogenase was partially purified starting from the cell-free extract of Lactobacillus plantarum 11 and the properties of the dehydrogenase were investigated. The intracellular activity of the enzyme was calculated. Using kinetic data concerning the dehydrogenase activity in the presence of various concentrations of coenzyme, and inorganic ions, the activity of this enzyme under the conditions thought to be comparable with an intracytoplasmic environment was calculated to be 0.04μ moles per mg dry weight of cells per hr. This value was 1% of lactic acid forming activity of the intact cells. A similar quantitative relationship was also demonstrated between C14O2 and lactate which were produced from 1-C14-glucose by washed cells. The role of the dehydrogenase in the cells of this organism was discussed with respect to its relation to the homo-lactic acid fermentation.

On the Metabolism of Organic Acids byClostridium acetobutylicum

Authors confirmatively observed the phenomena that in adequate conditions, the mode of acetone-butanol fermentation could be converted into homo-lactic acid fermentation, and that the formation of “Racemiase” (lactic acid racemiase) was intensified through the appropriate lactate level in growing culture of the bacterium, Clostridium acetobutylicum.Furthermore in this series of the papers, the possible role of “Racemiase” in the lactic acid metabolizing sequence was suggested, and the chemical properties of “Racemiase” as well as the mechanism of enzymatic racemization of lactic acid were investigated. The metabolism of several organic acids were also discussed in connection with the fermentation pathway of this organism.