Production of lactic acid from date juice extract with free cells of single and mixed cultures of Lactobacillus casei and Lactococcus lactis

The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3 g l⁻¹) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46 g l⁻¹, respectively. In the case of single Lactobacillus casei or Lactococcus lactis, the total percentage of glucose and fructose utilized were 82.2; 94.4% and 93.8; 60.3%, respectively, whereas in the case of mixed culture, the total percentage of glucose and fructose were 96 and 100%, respectively. These results showed that the mixed culture system gave better results than single cultures regarding lactic acid concentration, and sugar consumption.     

Limitation of growth and lactic acid production in batch and continuous cultures of Lactobacillus helveticus

Summary Continuous and batch cultures of Lactobacillus helveticus operated under different conditions were studied with respect to the limitation of growth and lactic acid production by increasing undissociated lactic acid and hydrogen ion concentrations, respectively. In a single-stage continuous culture without pH control a final pH of 3.8 and 65 mm undissociated lactic acid was obtained. In two-stage continuous cultures provided with different growth media and run at different pH values, 65–70 mm free acid was obtained in the second stage. Further batch-culture experiments showed growth limitation at 60–70 mm lactic acid. After growth ceased, production of lactate continued until a lactic acid concentration of about 100 mm was reached; obviously an uncoupling of growth and acid production had occurred. Examining the effect of different concentrations of either lactic acid or hydrochloric acid, added to growing batch cultures of L. helveticus, it was shown that the undissociated lactic acid concentration was responsible for growth limitation and lactic acid production in this organism, whereas the pH value had only an indirect effect.     

Lactic acid fermentation on barley flour without additional nutrients

Summary An amylolytic lactic acid producing Lactobacillus amylovorus produced 36 g/l of lactic acid in mixed cultures with L. casei without additional nutrients at 37 °C in 48 h, when barley flour concentration was 180 g/l (appr. 108 g/l starch) and barley malt quantity 0.8% of flour weight. This represented an improvement of up to 20% in comparison to the fermentation with L. amylovorus or L. casei alone. By simultaneous glucoamylase addition lactic acid production yield was about doubled. With L. casei the lactic acid yield was from 580 g in 72 h to 667 g in 144 h per kg barley flour.     

d(−)lactic acid production by suspended and aggregated continuous cultures of Bacillus laevolacticus

Summary A new method of d(–)lactic acid production based on the aggregated form of growth of Bacillus laevolacticus in continuous culture in an anaerobic gaslift reactor is presented. With glucose as the substrate a bacterial dry weight of 25 g·1^–1 and a lactic acid production rate of 13 g·1^–1·h^–1 was attained. In conventional glucose-limited chemostat cultures elevated levels of lactic acid stimulated specific lactic acid production while the formation rates of other end-products remained unaffected. In glucose-limited aggregated cultures lactic acid positively influenced the aggregation of cells, improving the volumetric production rate. It is concluded that lactic acid itself is a positive effector in the optimisation of lactic acid production with aggregated cultures of B. laevolacticus.Offprint requests to: J. P. de Boer     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

Kinetic study on pH dependence of growth and death of Streptococcus faecalis

A chemostat culture was used for lactic acid fermentation with Streptococcus faecalis at various pH values (8.0, 7.0, 6.0, 5.5, 5.0) and glucose concentrations (10, 20, 30 g/l). At every pH value, the reciprocals of the specific consumption rate of glucose and the specific production rate of lactic acid were linearly correlated to the reciprocal of the specific growth rate. The product, lactic acid, caused non-competitive inhibition of the specific growth rate at every pH value. Moreover, it was found that the cell death rate was dependent on pH and lactic acid. The death rate was smallest at pH 7.0 and increased with increasing lactic acid concentration. The kinetic equations of growth and death are proposed in a broader pH range. Correspondence to: H. Ohara     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium

Specific growth rates (μ) of two strains of Saccharomyces cerevisiae decreased exponentially (R ²>0.9) as the concentrations of acetic acid or lactic acid were increased in minimal media at 30°C. Moreover, the length of the lag phase of each growth curve (h) increased exponentially as increasing concentrations of acetic or lactic acid were added to the media. The minimum inhibitory concentration (MIC) of acetic acid for yeast growth was 0.6% w/v (100 mM) and that of lactic acid was 2.5% w/v (278 mM) for both strains of yeast. However, acetic acid at concentrations as low as 0.05–0.1% w/v and lactic acid at concentrations of 0.2–0.8% w/v begin to stress the yeasts as seen by reduced growth rates and decreased rates of glucose consumption and ethanol production as the concentration of acetic or lactic acid in the media was raised. In the presence of increasing acetic acid, all the glucose in the medium was eventually consumed even though the rates of consumption differed. However, this was not observed in the presence of increasing lactic acid where glucose consumption was extremely protracted even at a concentration of 0.6% w/v (66 mM). A response surface central composite design was used to evaluate the interaction between acetic and lactic acids on the specific growth rate of both yeast strains at 30C. The data were analysed using the General Linear Models (GLM) procedure. From the analysis, the interaction between acetic acid and lactic acid was statistically significant (P≤0.001), i.e., the inhibitory effect of the two acids present together in a medium is highly synergistic. Journal of Industrial Microbiology & Biotechnology (2001) 26, 171–177. Received 06 June 2000/ Accepted in revised form 21 September 2000     

Bioconversion of ovine scotta into lactic acid with pure and mixed cultures of lactic acid bacteria

Scotta is the main by-product in the making of ricotta cheese. It is widely produced in southern Europe and particularly in Italy where it represents a serious environmental pollutant due to its high lactose content. With the aim of evaluating whether scotta bioconversion into lactic acid can be considered as an alternative to its disposal, besides providing it with an added value, here the growth, fermentative performances, and lactic acid productions of pure and mixed cultures of Lactobacillus casei, Lactobacillus helveticus, and Streptococcus thermophilus were evaluated on ovine scotta-based media, without and with the addition of nutritional supplements. The outcomes indicate that ovine scotta can be utilized for the biotechnological production of lactic acid with yields up to 92%, comparable to those obtained on cheese-whey. Indeed, the addition of nutritional supplements generally improves the fermentative performances of lactic acid bacteria leading to about 2 g l⁻¹ h⁻¹ of lactic acid. Moreover, the use of mixed cultures for scotta bioconversion reduces the need for nutritional supplements, with no detrimental effects on the productive parameters compared to pure cultures. Finally, by using L. casei and S. thermophilus in pure and mixed cultures, up to 99% optically pure l-lactic acid can be obtained.     

Antimicrobial activity of lactobacilli and streptococci

Of nine strains of lactic acid bacteria commonly used as starter cultures for the dalry industry and ensiling, six (Lactobacillus bulgaricus, L. casei, L. acidophilus CH=5, L. plantarum, Streptococcus latis and Strep. taecium) had antibiotic activity. Gram-positive bacteria were more sensitive than Gram-negative bacteria to the antibiotics. The most sensitive strain of Staphylococcus aureus was used as a target micro-organism for the characterization of the antimicrobial substance. The cultures of Streptococcus faecium and L. plantarum gave the most intense antimicrobial activity. Adding CaCO₃ to the medium (to bind accumulated lactic acid) increased the antibiotic activity of the lactic acid bacteria.The authors are with the Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 41000 Zagreb, Yugoslavia.     

Enhancement of growth rate and β-galactosidase activity, and variation in organic acid profile of Bifidobacterium animalis subsp. lactis Bb 12

The behavior of Bifidobacterium animalis subsp. lactis Bb 12 under batch cultivation, after continuous culturing for up to 12 d, was monitored in skim milk-based media. Previous continuous culture for longer than 6 d affected the physiology of said microorganism. The minimum inhibitory concentrations of lactic and acetic acids increased from 18 to 26 g/l, whereas the molar ratio of acetic to lactic acid increased from 0.8 to 1.55, when the previous continuous culture increased its duration from 1 to 12 d. The specific lactose consumption rate decreased from 0.94 to 0.77 g_lactose/g_{cell dry mass}/h within the batch culture timeframe; this was concomitant with greater amounts of acetic and formic acids, and lower amounts of lactic acid produced. The β-galactosidase activity increased as continuous culturing time increased, and reached 446 units/ml by 12 d; however, the rate of enzyme synthesis decreased concomitantly. Succinic acid was produced during the exponential growth and stationary phases of the batch culture, but the former at exponential growth phase was higher as the continuous culturing time was longer. For comparison purposes, batch cultivation of samples taken from continuous cultures by 1 and 12 d was done using a semi-synthetic medium with glucose as carbon source; a pattern similar to that observed when using skim milk-based media was observed.     

The effect of lactic acid on anaerobic carbon or nitrogen limited chemostat cultures of Saccharomyces cerevisiae

Weak organic acids are well-known metabolic effectors in yeast and other micro-organisms. High concentrations of lactic acid due to infection of lactic acid bacteria often occurs in combination with growth under nutrient-limiting conditions in industrial yeast fermentations. The effects of lactic acid on growth and product formation of Saccharomyces cerevisiae were studied, with cells growing under carbon- or nitrogen-limiting conditions in anaerobic chemostat cultures (D=0.1 h⁻¹) at pH values 3.25 and 5. It was shown that lactic acid in industrially relevant concentrations had a rather limited effect on the metabolism of S. cerevisiae. However, there was an effect on the energetic status of the cells, i.e. lactic acid addition provoked a reduction in the adenosine triphosphate (ATP) content of the cells. The decrease in ATP was not accompanied by a significant increase in the adenosine monophosphate levels.     

Glucose and lactic acid trends in suspension cultures of two established mammalian cell strains in chemically defined media

In replicated 30 to 40-ml suspension cultures of rapidly proliferating monkey kidney cells of a comparatively fragile strain, the rates of glucose utilization and lactic acid accumulation averaged about 400 micrograms and 110 micrograms per 10⁶ cells per day respectively, with average molar La/Gl ratios of 0.48. These two rates of glucose utilization and lactic acid accumulation were about 4 × and 10 × as high as the corresponding rates in comparable cultures of the hardier strain 2071-L mouse fibroblasts under the same conditions, with average molar La/Gl ratios of 0.16. In comparable but nonproliferating suspension cultures of the same strain of monkey kidney cells, during about 3 weeks the rates were extremely high, with about 710 micrograms glucose utilized and 445 micrograms lactic acid accumulated per 10⁶ cells per day, with average molar La/Gl ratios of 1.37. The rates of glucose uptake and lactic acid accumulation were higher in the nonproliferating cultures aerated with 5% CO₂ in air than in those aerated with 10% CO₂ in air. This difference was associated with pH, which was higher in the former group. It was concluded that with this fragile strain of monkey Kidney cells(1) in nonproliferating cultures the cells were metabolizing actively but with a marked tendency to higher La/Gl ratios, (2) in the proliferating cultures the high rates of glucose utilization and lactic acid accumulation were definitely not directly correlated with the rate of growth, and (3) in none of the cultures was the amount of glucose remaining in the fluid at fluid changes so low as to have been a limiting factor. Information in the literature concering glucose utilization and lactic acid production by cells vitro is voluminous and in some respects contradictory. In the present study the rates were unexpectedly high for the monkey kidney cells, particularly those in the otherwise apparently inactive nonproliferating cultures. The data seem to be unique, in that an established strain of cells in chemically defined medium in suspension cultures has been characterized for these metabolic parameters in both proliferating cultures and in equivalent nonproliferating cultures under directly comparable conditions. The concept was developed that since these monkey kidney cells are obviously more fragile than the other cells examined, the complex physical stresses imposed upon these cells in agitated cultures can be modified and lessened in order to permit growth. Lessening of such mechanical stress waa brought about in several ways, of which only the smaller flask size seemed to be at least partly effective. Increasing either the concentration or the viscosity type of Methocel waa not effective.     

Acid Sensitivity of a Mutant of Lactococcus lactis subsp. lactis C2 with Reduced Membrane-bound ATPase Activity

Mutants with reduced membrane-bound ATPase activities were isolated from Lactococcus lactis subsp. lactis C2 as spontaneous neomycin-resistant mutants. Characteristics of the representative mutant, No. 1016–51, were compared with the parental strain in cultures using a jar fermentor with the pH controlled at various values. At pH 6.5, the fermentation patterns, i.e., glucose consumption, growth, and lactic acid production, of both strains appeared identical. At pH 4.5, however, the levels of growth, lactic acid production, and the amounts of lactic acid produced per cell after the culture for 24 h decreased to 60, 36, and 60% of the parental strain, respectively. During the cultures at pH 6.5, no differences were found in viabilities between both strains even after 80 h. On the other hand, at pH 4.0, the viable count of the strain No. 1016–51 in a 72-h culture decreased to less than 1% of that of the zero time, while the parental strain maintained its original viability. Therefore, it was concluded that the membrane-bound ATPase is essential for this organism to survive at low pH, probably through its function of proton pumping for maintaining cytoplasmic pH levels.     

Lactic acid production by aggregated continuous cultures of Lactobacillus acidophilus under aerobic conditions

Summary The ability of Lactobacillus acidophilus to aggregate, to produce lactic acid for a long term continuous fermentation process and to exist as aggregate cell cultures in a gas-lift reactor under aerobic conditions was studied. The main product of fermentation was lactic acid and only the traces of other end-products were determined. The highest fermentation efficiency of lactic acid was 98.6% and the highest productivity was 9.6 g.l^–1.h^–1 of lactic acid.     

Effect of oxygen on batch yogurt cultures

A yogurt culture (Streptococcus thermophilus 15HA + Lactobacillus delbrueckii subsp. bulgaricus 2-11) was studied in conditions of aerobic batch fermentation (10–40% dissolved oxygen in milk). The growth and acidification of S. thermophilus 15HA were stimulated at 20% oxygen concentration and the lactic acid process in a mixed culture was shortened by 1 h (2.5 h for the aerobic culture and 3.5 h for the anaerobic mixed culture). Streptococcus thermophilus 15HA oxygen tolerance was significantly impaired at oxygen concentrations in the milk above 30%. Though S. thermophilus 15HA was able to overcome to some extent the impact of high oxygen concentration (40%), the lactic acid produced was insufficient to coagulate the milk casein (4.0 g lactic acid l⁻¹ in the mixed culture and 3.8 g lactic acid l⁻¹ in the pure culture). A dramatic decrease in the viable cell count of L. delbrueckii subsp. bulgaricus 2-11 in the pure and mixed cultures was recorded at 30% dissolved oxygen. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lactic acid production from xylose by the fungus Rhizopus oryzae

Lignocellulosic biomass is considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentation of bulk chemicals such as lactic acid. The filamentous fungus Rhizopus oryzae is able to grow in mineral medium with glucose as sole carbon source and to produce optically pure l(+)-lactic acid. Less is known about the conversion by R. oryzae of pentose sugars such as xylose, which is abundantly present in lignocellulosic hydrolysates. This paper describes the conversion of xylose in synthetic media into lactic acid by ten R. oryzae strains resulting in yields between 0.41 and 0.71 g g⁻¹. By-products were fungal biomass, xylitol, glycerol, ethanol and carbon dioxide. The growth of R. oryzae CBS 112.07 in media with initial xylose concentrations above 40 g l⁻¹ showed inhibition of substrate consumption and lactic acid production rates. In case of mixed substrates, diauxic growth was observed where consumption of glucose and xylose occurred subsequently. Sugar consumption rate and lactic acid production rate were significantly higher during glucose consumption phase compared to xylose consumption phase. Available xylose (10.3 g l⁻¹) and glucose (19.2 g l⁻¹) present in a mild-temperature alkaline treated wheat straw hydrolysate was converted subsequently by R. oryzae with rates of 2.2 g glucose l⁻¹ h⁻¹ and 0.5 g xylose l⁻¹ h⁻¹. This resulted mainly into the product lactic acid (6.8 g l⁻¹) and ethanol (5.7 g l⁻¹).     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from a mixture of xylose and glucose by co-cultivation of lactic acid bacteria

The production of optically pure lactic acid in a high yield from xylose or a mixture of xylose and glucose, which is a model hydrolysate of lignocellulose, is described. In a single cultivation, Enterococcus casseliflavus produced 38 g/l of lactic acid with an optical purity of 96% enantiomeric excess (ee) and 6.4 g/l of acetic acid from 50 g/l of xylose when MRS medium was used. When a mixture of 50 g/l of xylose and 100 g/l of glucose was used as the carbon source in a cultivation of E. casseliflavus alone, glucose was converted to lactic acid in the early phase of the cultivation but xylose was hardly consumed. In a co-cultivation where E. casseliflavus and Lactobacillus casei specific for glucose were simultaneously inoculated, little or no lactic acid was produced after the glucose was almost consumed. A co-cultivation with two-stage inoculation (in which E. casseliflavus was added at a cultivation time of 40 h after L. casei cells were inoculated) resulted in complete consumption of 50 g/l of xylose and 100 g/l of glucose. In the co-cultivation, 95 g/l of lactic acid with a high optical purity of 96% ee was obtained at 192 h. Such a co-cultivation using two microorganisms specific for each sugar is considered to be one promising cultivation technique for the efficient production of lactic acid from a sugar mixture derived from lignocellulose.     

Effect of expressing polyhydroxybutyrate synthesis genes (phbCAB) in Streptococcus zooepidemicus on production of lactic acid and hyaluronic acid

Hyaluronic acid (HA) has been industrially produced using the gram-positive bacterium Streptococcus zooepidemicus. Large amount of lactic acid formation was one of the important factors that restricted cell growth and HA productivity and lowered the substrate to HA conversion efficiency in a fermentor. In this study, polyhydroxybutyrate (PHB) synthesis genes (phbCAB) of Ralstonia eutropha were cloned from the plasmid pBHR68 and were inserted into the plasmid pEU308, an expression vector for gram-positive bacteria. The plasmid was transformed into S. zooepidemicus by electroporation. β-Ketothiolase (PhbA), acetoacetyl-CoA reductase (PhbB), and polyhydroxyalkanoate (PHA) synthase (PhbC) activity assays were carried out to demonstrate the expression of these genes. The PhbA and PhbB activities were 3.13 and 1.23 U mg⁻¹, respectively. No PhbC activities were detected. In shake flask studies, there was no obvious difference between the wild-type and recombinant S. zooepidemicus harboring phbCAB genes in terms of lactic acid and HA formation. However, in fermentor studies, the recombinant produced only 40 g L⁻¹ lactic acid and 7.5 g L⁻¹ HA, whereas the wild type produced 65 g L⁻¹ lactic acid and 5.5 g L⁻¹ HA. These results suggested that expression of phbCAB genes in S. zooepidemicus could help regulate HA production metabolism. Because the lactic acid formation in S. zooepidemicus was sensitive to cellular oxidation/reduction potential, it is proposed that the PHB synthesis pathway could act as a regulator to adjust the cellular oxidation/reduction potential. This is the first study demonstrating that PHA synthesis related to energy and carbon metabolism could be employed as a pathway to regulate other cellular metabolism and possibly to regulate the production of other metabolic products.     

Lactic acid production from agriculture residues

Various agriculture feedstock residues were evaluated for lactic acid production by simultaneous saccharification and fermentation (SSF) using Lactobacillus delbrueckii and Lactobacillus plantarum, without any additional nutrients. Lactic acid production was higher in alfalfa fiber and soya fiber compared to corncob (soft) and wheat straw. In Lactobacillus plantarum, the amount of lactic acid obtained from alfalfa fiber and soya fiber was 46 and 44 g/100 g fiber, respectively. However, in Lactobacillus delbrueckii, the lactic acid production in soya fiber was 44 g/100 g fiber and that of alfalfa was 32 g/100 g fiber. Small amounts of acetic acid were also produced from SSF of agricultural feedstocks residues. During SSF of alfalfa fiber, lactic acid production in both L. delbrueckii and L. plantarum was enhanced by adding pectinases and cellulases. Lactic acid production from alfalfa fiber did not change with increasing O₂ transfer rates in the fermentation medium, whereas acetic acid production in both Lactobacillus cultures increased with increasing O₂ transfer rates.