Riboflavin Biosynthesis by Candida robusta

During the study on the oxidative sugar metabolism of yeasts, it was found that six strains of Candida robusta, isolated from fruits, produced large amounts of yellow pigment in shaking culture and this pigment was identified as riboflavin. Riboflavin production by C. robusta has never been reported.

Some notable characteristics of C. robusta in riboflavin production were found. As nitrogen sources, ammonium salts and urea were favorable, but nitrate and organic nitrogen sources such as glycine, asparagine and peptone were not utilized for riboflavin production. Riboflavin was not produced in still culture; a highly aerobic condition, as may be obtained by shaking culture, wa, essential. The addition of excess CaCO₃ was also necessary. Acetic acid, added as the Ca salt in its production as a sole carbon source, was more effective than sugars and optimum concentration of this acid was 7%. Riboflavin were obtained in yields as high as 32 to 34 mg % from the acetate medium after 8 days.     

Optimization of riboflavin production by recombinant <Emphasis Type="Italic">Bacillus subtilis</Emphasis> RH44 using statistical designs

A sequential optimization strategy, based on statistical experimental designs, was used to enhance the production of riboflavin by recombinant Bacillus subtilis RH44. In the first instance, the medium components were optimized in shake flask cultures. After preliminary experiments of nitrogen source selection, the two-level Plackett–Burman (PB) design was implemented to screen medium components that significantly influence riboflavin production. Among the 15 variables tested, glucose, NaNO₃, K₂HPO₄, ZnSO₄, and MnCl₂ were identified as the most significant factors (confidence levels above 95%) for riboflavin production. The optimal values of these five variables were determined by response surface methodology (RSM) based on the central composite design (CCD). The validity of the model developed was verified, and the optimum medium led to a maximum riboflavin concentration of 6.65 g/l, which was 44.3 and 76.4% higher than the improved medium and the basal medium, respectively. A glucose-limited fed-batch culture profile in a 5-l fermentor was consequently designed according to the above optimum medium in shake flasks. A final riboflavin concentration of 16.36 g/l was obtained in 48 h, which further verified the practicability of this optimum strategy.     

Calcium carbonate precipitation by heterotrophic bacteria isolated from biofilms formed on deteriorated ignimbrite stones: influence of calcium on EPS production and biofilm formation by these isolates

Heterotrophic CaCO₃-precipitating bacteria were isolated from biofilms on deteriorated ignimbrites, siliceous acidic rocks, from Morelia Cathedral (Mexico) and identified as Enterobacter cancerogenus (22e), Bacillus sp. (32a) and Bacillus subtilis (52g). In solid medium, 22e and 32a precipitated calcite and vaterite while 52g produced calcite. Urease activity was detected in these isolates and CaCO₃ precipitation increased in the presence of urea in the liquid medium. In the presence of calcium, EPS production decreased in 22e and 32a and increased in 52g. Under laboratory conditions, ignimbrite colonization by these isolates only occurred in the presence of calcium and no CaCO₃ was precipitated. Calcium may therefore be important for biofilm formation on stones. The importance of the type of stone, here a siliceous stone, on biological colonization is emphasized. This calcium effect has not been reported on calcareous materials. The importance of the effect of calcium on EPS production and biofilm formation is discussed in relation to other applications of CaCO₃ precipitation by bacteria.     

Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation

Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B₂) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-l-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3–3.5 times higher as compared to the parental strain. It produced 50–70 times more riboflavin in iron sufficient synthetic media relative to the parental wild-type strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.     

Induction of Calcium Carbonate by Bacillus cereus

The purpose of this research was to study how the bacteria Bacillus cereus (DCB1) utilizes calcium ions in a culture medium with carbon dioxide (CO₂) to yield calcium carbonate (CaCO₃). The bacteria strain DCB1 was a dominant strain isolated from dolomitic surfaces in areas of Karst topographies. The experimental method was as follows: a modified beef extract-peptone medium (beef extract 3.0 g, peptone 10 g, NaCl 5.0 g, CaCl₂ 2.0 g, glass powder 2.0 g, distilled water 1 L, and a pH between 6.5 and 7.5) was inoculated with B. cereus to attempt to induce the synthesis of CaCO₃. The sample was then processed by centrifugation every 24 h during the 7-day cultivation period. The pH, carbonic anhydrase (CA) activity, and the concentrations of both HCO^- ₃ and Ca²⁺ in the supernatant fluid were measured. Subsequently, precipitation in the culture medium was analyzed to confirm, or otherwise, the presence and if present, the formation, of CaCO₃. Methods used included X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Energy Dispersive Spectroscopy (EDS). Meanwhile, the carbon source in the carbonate was classified by its isotope composition. Results showed that B. cereus can improve its pH value in this culture medium; concentrations of HCO^- ₃ and Ca²⁺ showed a significant decline over the duration of the cultivation period. CA activity reached its maximum during the second day; XRD, SEM, TEM, and isotope analysis all revealed the presence of CaCO₃ as a precipitate. Additionally, these results did not occur in an aseptic control group: no detectable level of CaCO₃ was produced therein. In conclusion: B. cereus can metabolize active materials, such as secretase, by its own growth and metabolism, and can either utilize atmospheric CO₂, or respire, to induce CaCO₃ production. Experimental evidence is offered for a concomitant CO₂ reduction and CaCO₃ induction by microorganisms.     

Effect of calcium carbonate and iron on the availability and uptake of iron,manganese, phosphorus and calcium in pea (Pisum sativum L.)

Summary The effect of CaCO₃ and iron on the availability of iron, manganese phosphorus and calcium was studied in the greenhouse on pea (Pisum sativum L.) crop on a light textured soil, which was marginal in exchangeable calcium. Addition of calcium carbonate caused significant increase in dry matter yield with no added iron at both the stages of crop growth. But yeild intended to decrease with 8% CaCO₃ at 75 days of crop growth. Dry matter yield also increased with the addition of iron upto 10 ppm at 45 days and upto 5 ppm at 75 days. The iron concentration and uptake decreased with the increase in CaCO₃ and increased with the application of iron at both the stages of crop growth. The application of iron and CaCO₃ decreased concentration and uptake of phosphorus significantly at both the stages.Like phosphorus, concentration and uptake of manganese also decreased with the increase in added CaCO₃ upto 8% and iron upto 20 ppm at 45 and 75 days. The concentration of calcium increased with the addition of CaCO₃ to the extent of 50 and 40% with 8% CaCO₃ at 45 and 75 days, while the uptake of calcium increased more than 3 folds at 45 days and more than 2 folds at 75 days. The concentration of calcium decreased with the application of iron upto 20 ppm but the uptake at 45 days increased upto 10 ppm and at 75 days upto 5 ppm and then decreased.The concentration of Fe, P and Ca decreased at 75 days and that of Mn increased while the uptake of all these nutrients increased at 2nd stage due to higher dry matter.     

Effect of calcium carbonate and organic matter on the growth and concentration of iron and manganese in sorghum (Sorghum bicolor)

Summary The effect of CaCO₃ and organic matter on the availability of iron and manganese was studied on sorghum (Sorghum bicolor). Addition of calcium carbonate caused significant decrease in dry matter and grain yield. But yields tended to increase with the addition of organic matter at the rate of 15 t/ha. The iron concentration decreased with the addition of CaCO₃ at all the stages of crop growth. Like iron, concentration of manganese also decreased with the increase in added CaCO₃. Organic matter increased the iron concentration both at seedling and flowering stage. The concentration of manganese was, however, dependent on the stage of the crop.     

An Alternative Biotechnological Tool for Magnesite Enrichment: Lactic Acid Bacteria Isolated from Soil

Abstract

Calcium carbonate (CaCO₃) is found in different polymorph structures such as aragonite, vaterite, and calcite. The most common and stable form of CaCO₃, calcite, which is abundant in sedimentary rocks as magnesite ore. Magnesite has application areas in many industrial fields including paper, pharmaceutical and refractory materials. Magnesite is theoretically formulated MgCO₃, but contains many impurities (silicium, iron, and also calcite), that limits its usability and applicability. In this research, we aimed to investigate the decalcification possibility of the raw magnesite material through application of Enterococcus feacelis (EF) with CaCO₃ dissolution ability. The exact mechanism of CaCO₃ dissolution was investigated by carbonic anhydrase enzyme assay and HPLC analysis of organic acids produced by EF. Consequently, EF reduced the amount of CaCO₃ from 2.94% to 0.49% which means a reduction (≈83.33%) in the rate of CaCO₃ percentage. As a result of the experiments, it was observed that different organic acids produced by bacteria reacted with CaCO₃ and removed the lime of magnesite ore. The bacteria used in the study did not show any pathogenic properties in rats, thus, it can be used safely for the industrial applications.     

Ferric iron and extracellular electron shuttling increase xylose utilization and butanol production during fermentation with multiple solventogenic bacteria

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild-type cells are goals for biofuel production. Xylose consumption, butanol production, and hydrogen production increased in both Clostridium beijerinckii and a novel solventogenic bacterium (strain DC-1) when anthraquinone-2,6,-disulfonate (AQDS) or riboflavin were used as redox mediators to transfer electrons to poorly crystalline Fe(OH)₃ as an extracellular electron sink. Strain DC-1 was most closely related to Rhizobiales bacterium Mfc52 based on 95% 16S rRNA gene sequence similarity, which demonstrates that this response is not limited to a single genus of xylose-fermenting bacteria. Xylose utilization and butanol production were negligible in control incubations containing cells plus 3% (w/v) xylose alone during a 10-day batch fermentation, for both strains tested (n-butanol titers of 0.05 g L⁻¹). Micromolar concentrations of AQDS and riboflavin were added as electron shuttling compounds with poorly crystalline Fe(OH)₃ as an insoluble electron acceptor, and respective n-butanol titers increased to 6.35 and 7.46 g L⁻¹. Increases in xylose consumption for the iron treatments were relatively high, from less than 0.49 g L⁻¹ (xylose alone, no iron or electron shuttling molecules) to 25.98 and 29.15 g L⁻¹ for the AQDS and riboflavin treatments, respectively. Hydrogen production was also 3.68 times greater for the AQDS treatment and 5.27 greater for the riboflavin treatment relative to controls. Strain DC-1 data were similar, again indicating that the effects are not specific to the genus Clostridium.

     

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes

Improved strains for the production of riboflavin (vitamin B₂) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5′-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l⁻¹ for 72 h in a 5-l jar fermentor without any end product inhibition. Received: 23 August 1999 / Received revision: 13 October 1999 / Accepted: 5 November 1999     

Production of exopolysaccharide from ethanol inAgrobacterium radiobacter

Agrobacterium radiobacter produces an extracellular polysaccharide from various carbon sources. The exopolysaccharide is produced from ethanol also in a minimal medium containing nitrate as the sole nitrogen source. On cultivation in a medium without CaCO₃ only a minute amount of ethanol is converted to the exopolysaccharide. Both ethanol and nitrate in higher concentrations inhibit the growth rate. In a medium with CaCO₃ the proportion of ethanol converted to the polysaccharide is about ten-fold higher and the inhibitory effect of ethanol and nitrate on the growth rate is analogous to that found in the medium without CaCO₃-Comparison of results of mass and electron balance with a kinetic model shows that the parameters obtained in cultivations with CaCO₃ are less reliable. The balances point to the possibility of formation of other extracellular products.     

Importance of B4 Medium in Determining Organomineralization Potential of Bacterial Environmental Isolates

B4 precipitation medium has been used as the preferred medium for studying mineral precipitation using bacterial strains in vitro since pioneer studies were performed by Boquet and coworkers in 1973. Using this medium, several authors have demonstrated that some environmental isolates were able to precipitate minerals, yet others did not. The main goal of the current study is to understand whether pH and buffer conditions would have a significant effect on mineral precipitation results for environmental isolates grown on B4. For this study, a total of 49 strains isolated from natural environments from Puerto Rico were grown on B4 plates, and their CaCO₃ precipitation potential was investigated. Our findings revealed a strong correlation between a lack of CaCO₃ precipitation and the acidification of the B4 plates by the colonies. The ability to precipitate CaCO₃ could be restored by buffering the B4 medium to a pH of 8.2. Buffering capacity of the medium was proposed to be involved in CaCO₃ precipitation: acid-base titrations conducted on the individual ingredients of B4 showed that yeast extract has a poor buffering capacity between pH 6.5–7.5. This pH range corresponds to the pH of B4 plates [6.87 (±0.05)] prior to the inoculation. This might explain why B4 is such a good precipitation medium: a small variation in the H⁺/OH^? balance during microbial growth and precipitation produces rapid changes in the pH of the medium. Finally, an amorphous matrix was distributed within 90% of the examined crystals generated on B4 medium by the environmental strains. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental file.     

Effect of cultivation conditions on the activity of the beromycin producerStreptomyces glomeratus 3980 and its spontaneous variants

Optimal conditions for the submerged cultivation ofStreptomyces glomeratus 3980, producer of the anthracycline antibiotics beromycins, and its variants were sought in media with glucose, soybean meal and salts differing in the content of ammonium sulphate. As compared with the original activity of the strain the antibiotic titre of some variants increased about 12 times on increasing the glucose concentration from 3 to 5 %, or on omitting CaCO₃ from the medium (i.e. under conditions leading to an increased production of propionic acid and suppression of production of the melanin-like pigment). In melanin-less variants accumulating propionate even under standard conditions the activity increased about 18–40 times in the medium with 3 % glucose and 0.2 % CaCO₃ under conditions of more intensive aeration (i.e. under conditions when no propionic acid accumulated). Individual strains also differed in the requirement for (NH₄)₂SO₄ in the medium, their response to changes of volume of the vegetative inoculum and sensitivity to MgSO₄.7H₂O. The biosyiithetic activity of all strains was inhibited by the addition of ZnSO₄.7H₂O or CaCl₂ and substitution of glucose with starch, lactose or sucrose.     

Isolation of an oxalate-resistant Ashbya gossypii strain and its improved riboflavin production

An oxalate-resistant strain of Ashbya gossypii was naturally isolated from spores grown on an oxalate-containing medium, and its medium was optimized to improve riboflavin production. Riboflavin production by the resistant strain was three-fold higher than that by the wild-type organism when grown in flask cultures. Medium optimization increased the riboflavin production by the resistant strain to 5 g l⁻¹, which was five-fold higher than that obtained by the wild-type strain. The productivity was reproduced in a 3-l bioreactor. During the early growth phase, the specific activity of isocitrate lyase in the oxalate-resistant strain was slightly higher than that in the wild-type strain. Proteomic analysis of the oxalate-resistant strain revealed that the expression of aldose reductase and cobalamin-independent methionine synthase decreased significantly. This is the first report that describes the natural isolation of a riboflavin producer using an antimetabolite-containing medium to enhance the riboflavin production level. This method should also be useful for improving the productivity of other bioproducts since it does not require any mutations or genetic modifications of the microorganism.     

Involvement of etfA gene during CaCO3 precipitation in Bacillus subtilis biofilm

The eftA gene in Bacillus subtilis has been suggested to be involved in the oxidation/reduction reactions during fatty acid metabolism. Interestingly etfA deletion in B. subtilis results in impairment in CaCO₃ precipitation on the biofilm. Comparisons between the wild type B. subtilis 168 and its etfA mutant during in vitro CaCO₃ crystal precipitation (calcite) revealed changes in phospholipids membrane composition with accumulation of up to 10% of anteiso-C_17:0 and 11% iso-C_17:0 long fatty acids. Ca²⁺ nucleation sites such as dipicolinic acid and teichoic acids seem to contribute to the CaCO₃ precipitation. etfA mutant strain showed up to 40% less dipicolinic acid accumulation compared with B. subtilis 168, while a B. subtilis mutant impaired in teichoic acids synthesis was unable to precipitate CaCO₃. In addition, B. subtilis etfA mutant exhibited acidity production leading to atypical flagella formation and inducing extensive lateral growth on the biofilm when grown on 1.4% agar. From the ecological point of view, this study shows a number of physiological aspects that are involved in CaCO₃ organomineralization on biofilms.     

Enhanced curdlan production in <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749 by addition of low-polyphosphates

A large amount of adenosine triphosphate with high energy phosphate bonds is required for uridine triphosphate regeneration during curdlan biosynthesis by Agrobacterium sp. ATCC 31749. To supply high energy for curdlan synthesis, three low-polyphosphates (Na₄P₂O₇, Na₅P₃O₁₀, and (NaPO₃)₆) with higher energy phosphate bonds were employed to substitute for KH₂PO₄-K₂HPO₄ in fermentation medium. Two genes encoding the polyphosphate metabolizing enzymes, polyphosphate kinase and exopolyphosphatase, were amplified and showed 95% homology to those in Agrobacterium sp. C58 by sequence analysis. The curdlan yields were enhanced by 23 and 134% when phosphate concentrations 0.024 mol/L of Na₅P₃O₁₀ and 0.048 mol/L of (NaPO₃)₆ respectively, were added in the medium. The maximum curdlan yield of 30 ± 1.02 g/L was obtained with the addition of 0.048 mol/L of (NaPO₃)₆ with 5 g/L CaCO₃ in the medium. When CaCO₃ was removed from the culture and the three lowpolyphosphates were added, the pH and biomass yield dropped remarkably and little or no curdlan was produced. The culture containing 0.048 mol/L of (NaPO₃)₆ was mixed with KH₂PO₄-K₂HPO₄ and CaCO₃ in the medium, but showed no effect on curdlan production. However, curdlan yield was improved by 49 ∼ 60% when CaCO₃ was removed from the medium and KH₂PO₄-K₂HPO₄ acted as a buffer. It appears that the positive effect of (NaPO₃)₆ on curdlan production required the buffering capacity of CaCO₃ and the absence of KH₂PO₄-K₂HPO₄ competing as a phosphate supplier.     

Helicobacter pylori ribBA-Mediated Riboflavin Production Is Involved in Iron Acquisition

In this study, we cloned and sequenced a DNA fragment from an ordered cosmid library of Helicobacter pylori NCTC 11638 which confers to a siderophore synthesis mutant of Escherichia coli (EB53 aroB hemA) the ability to grow on iron-restrictive media and to reduce ferric iron. Sequence analysis of the DNA fragment revealed the presence of an open reading frame with high homology to the ribA gene of Bacillus subtilis. This gene encodes a bifunctional enzyme with the activities of both 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase and GTP cyclohydrolase II, which catalyze two essential steps in riboflavin biosynthesis. Expression of the gene (designated ribBA) resulted in the formation of one translational product, which was able to complement both the ribA and the ribB mutation in E. coli. Expression of ribBA was iron regulated, as was suggested by the presence of a putative FUR box in its promotor region and as shown by RNA dot blot analysis. Furthermore, we showed that production of riboflavin in H. pylori cells is iron regulated. E. coli EB53 containing the plasmid with H. pylori ribBA excreted riboflavin in the culture medium, and this riboflavin excretion also appeared to be iron regulated. We postulate that the iron-regulated production of riboflavin and ferric-iron-reduction activity by E. coli EB53 transformed with the H. pylori ribBA gene is responsible for the survival of EB53 on iron-restrictive medium. Because disruption of ribBA in H. pylori eliminates its ferric-iron-reduction activity, we conclude that ribBA has an important role in ferric-iron reduction and iron acquisition by H. pylori.     

Physiological restriction of the L-lactic acid production by Rhizopus arrhizus

The influence of basic physiological factors on the quality of inocula and L(+)-lactic acid production by Rhizopus arrhizus CCM 81 09 were studied. The most effective preparation of the spores (5 × 10⁷ spores/ml) and subsequent good lactate production was achieved on the agar medium with soil extract and malt agar. The optimum initial amount of active spores for inoculation was 10³–10⁴ spores/ml. The preparation of inoculum required intensive stirring with lower aeration and pH maintained in the range from 4.8 to 6.0 by the addition of CaCO₃. The maximum yield of lactic acid production was achieved by using 5% (v/v) of 24-h-old inoculum. The intensity of lactic acid production in the inoculum was proportional to its production in the subsequent steps of fermentation and can be used as a fast control of the physiological state of the producers.     

Growth inhibition ofMicrococcus pyogenes by manganese and riboflavin

Summary Riboflavin, in the presence of manganese, inhibited the growth ofMicrococcus pyogenes var.aureus. The inhibitory action was shown to be dependent upon aerobiosis, and was characterized predominately as a prolongation of the lag phase. Increasing concentrations of either manaanese or riboflavin, in the presence of a constant quantity of the other, enhanced the inhibitory effect. Ultra-violet irradiation of riboflavin reduced its toxic capacity in proportion to the amount of riboflavin degraded. Riboflavin-5-phosphate was equally as inhibitory as riboflavin. Ferrous iron was capable of a non-stoichiometric antagonism at low concentrations of riboflavin and/or manganese. Suported, in part, by a research grant (E-605) from the National Institutes of Health, U.S. Public Health Service. A preliminary report has appeared (Ramsey andWilson, 1956).     

Optimization of lactic acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation was investigated. Shake flasks proved to be a better fermentation system for this purpose. Substitution of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid concentration was achieved without treatment of molasses. A Central Composite Design was employed to determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast extract, CaCO₃). A satisfactory fit of the model was realized. Lactic acid production was significantly affected both by sucrose–yeast extract and sucrose–CaCO₃ interactions, as well as by the negative quadratic effects of these variables. Sucrose and yeast extract had a linear effect on lactic acid production while the CaCO₃ had no significant linear effect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast extract and CaCO₃ of 89.93, 45.71 and 59.95 g/l, respectively.