Nitrogen fixation by Bacillus strains isolated from the rhizosphere of Ammophila arenaria

Summary Several Bacillus strains, from the rhizosphere of Ammophila arenaria, appeared on ‘nitrogen-free’ agar plates. They were able to grow in nitrogen-poor medium to which 0.1% yeast extract was added. Three of these bacilli were tested for their ability to fix nitrogen using the acetylene reduction assay. The C₂H₂-reducing activity was determined at 8-hour intervals during their growth cycle. C₂H₂ reduction (and accordingly N₂ fixation) was greater under anaerobic than aerobic conditions. Additions of 0.1% CaCO₃ significantly increased the C₂H₂-reducing activity under both conditions. Characterisation suggests that these strains are new nitrogen-fixing Bacillus species. re]19740121     

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.     

Organic Acid Production by Basidiomycetes: III. Cultural Conditions for L-Malic Acid Production

Cultural conditions were examined for the purpose of increasing yields of l-malic acid by the Basidiomycetes Schizophyllum commune and Merulius tremellosus, which have the ability to produce this acid as a main product in CaCO₃-containing medium in shaken culture. The most favorable nitrogen sources selected were 0.3% (NH₄)₂SO₄ and 0.18% NH₄Cl. Effective combinations of inorganic salts in the medium were 0.1% KH₂PO₄, 0.05% MgSO₄·7H₂O, and 0.05% KCl, and suitable concentrations of glucose were 5 to 10%. Several nonionic surface-active agents promoted the filamentous mycelial growth of these strains and increased acid production. In particular, Tween 80 in 0.3% concentration markedly stimulated malic acid production by S. commune, and yields greater than 50% based on available glucose, were obtained after 10 to 14 days. Acid production by M. tremellosus was stimulated most with 0.5% Carbowax 4000 (polyethylene glycol), and the resultant yields were more than 40%.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).     

Yield of submerged paddy and uptake of Zn,P and N as affected by liming and Zn fertilizers

The effects of CaCO₃, Zn sources and levels on the yield of submerged paddy and uptake of Zn, P and N to paddy were studied in green-house at Haryana Agricultural University, Hissar. Powdered CaCO₃ was mixed at 0,4 and 8 per cent and Zn was added at 0,5 and 10 ppm through ZnSO₄.7H₂O, ZnO and Zn EDTA separately. Dry weight at tillering and heading and grain and straw at maturity decreased significantly with 4 and 8 per cent CaCO₃ in comparison to the control. Increasing Zn application increased the dry weight and grain yield. Zn EDTA gave highest yield of paddy followed by ZnSO₄.7H₂O and ZnO.Increasing the application of CaCO₃ from 0–8 per cent decreased the concentration and uptake of Zn and increasing Zn application from 0–10 ppm increased concentration and uptake of Zn in paddy at tillering, heading and maturity. Zn EDTA gave the highest concentration and uptake of Zn followed by ZnSO₄.7H₂O and ZnO. There was interaction between Zn sources and CaCO₃.The concentration and uptake of N and P in paddy dry matter at tillering and heading and straw and grain at maturity decreased as compared to control with increasing CaCO₃ addition. The concentration and uptake of N increased and that of P decreased in paddy dry matter straw and grain with increasing Zn application. The highest concentration of N was observed with ZnO, followed by ZnSO₄.7H₂O and Zn EDTA. But highest uptake of N was observed with Zn EDTA followed by ZnSO₄.7H₂O and ZnO. As regards concentration and uptake of P, it was highest with ZnO followed by ZnSO₄.7H₂O and Zn EDTA.     

Ketogluconate formation by Gluconobacter species

Summary When G. oxydans ATCC 621-H was grown in batch culture in a complex medium with glucose, ketogluconates were produced when the pH in the culture was maintained at 5.5. Without pH control gluconate was the only product of glucose oxidation, but at pH 5.5 the gluconate so produced was further oxidized to ketogluconates. Production of ketogluconates started when glucose was almost completely exhausted. It was shown that the actual glucose and gluconate concentrations in the culture do not determine the onset of ketogluconate formation during growth. Both 2 and 5 ketogluconate were produced. Addition of CaCO₃ to the medium favored the production of 5 ketogluconate. However, under these conditions minor quantities of 2 ketogluconate were also formed. The sequential production of gluconate and ketogluconates from glucose was not only restricted to G. oxydans ATCC 621-H. A number of G. oxydans strains when grown under standard conditions in a pH controlled batch culture, all produced ketogluconates from glucose via an intermediate accumulation of gluconate. Although the ratios of the ketogluconates produced varied from strain to strain, all strains produced both 2 and 5 ketogluconate.     

Factors determining the degree of anaerobiosis of Bifidobacterium strains

Summary The degree of sensitivity of twelve Bifidobacterium (Lactobacillus bifidus) strains to O₂ was determined by measuring the size of the inhibition zones obtained when the bacteria were grown in deep agar cultures under air, and by measuring growth in aerated cultures. The size of the inhibition zones varied from 1 to 23 mm. Growth in aerated cultures differed markedly for the strains investigated. No strain grew on agar plates under aerobic conditions.The small inhibition zone of three Bifidobacterium strains might be explained by the presence of a weak catalase activity, which removes traces of H₂O₂ possibly formed. It is also possible that the NADH oxidase of these strains does not form H₂O₂ at all. Most probably, the lack of growth on an agar medium results from the fact that these strains only grow below a certain oxidation-reduction potential.One strain, which was rather insensitive to O₂, formed a small amount of H₂O₂ from NADH oxidation. The absence of H₂O₂ in aerated liquid cultures and cell suspensions of this strain, which lacked catalase and NAD peroxidase activity, must be explained by removal of the traces of H₂O₂ formed, by an unknown peroxidase system or by a chemical reaction with pyruvate formed during glucose fermentation.For two strains, which were moderately sensitive to O₂, accumulation of H₂O₂ seems to be the principal reason for anaerobiosis. H₂O₂ turned out to inactivate specifically fructose-6-phosphate phosphoketolase, a key enzyme of the fermentation pathway of bifidobacteria.In the culture medium of two strains, which were extremely sensitive to O₂, no H₂O₂ could be detected after aeration. During anaerobic growth of these strains, the oxidation-reduction potential of the culture decreased so much that neutral red was decolourized. Cell suspensions of these strains only fermented glucose when cysteine was added. It was concluded that these strains required a low oxidation-reduction potential for growth and fermentation.     

Gluconic Acid Fermentation by Pullularia pullulans

During the study on the sugar metabolism of molds, several strains of Pullularia pullulans were found to produce large amounts of gluconic acid from glucose. Thirty seven strains of P. pullulans were then tested for their acid-producing abilities. Seven strains did not produce any amount of gluconic acid. However, all of the other strains were shown to be capable of producing this acid. The superior strains produced yiclds of gluconic acid as high as about 90%, based on glucose available, in shaking cultures at 30°C after 2 days. The yields were increased up to approximately 100% during later stages. In addition to high yields, gluconic acid was produced exclusively by these strains. Glutamic acid and inorganic ammonium salts, such as (NH₄)₂SO₄, NH₄Cl and (NH₄)₂HPO₄, were favorable nitrogen sources for acid production. In the case of (NH₄)₂SO₄, the optimum concentration was 0.05%. The addition of CaCO₃ was essential for gluconic acid production by P. pullulans and a 3% concentration of CaC0₃ appeared to be desirable for the maximum conversion to gluconic acid in a medium containing 10% glucose.     

A new medium for spore production of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> using response surface methodology

Optimization of the medium components which enhance sporulation of the two mating types of the fungus Blakeslea trispora ATCC 14271 and ATCC 14272 (a heterothallic Zygomycota producing carotene) was achieved with the aid of response surface methodology (RSM). Glucose, corn steep liquor, yeast extract, and ammonium sulfate were investigated as carbon and nitrogen sources in a basal medium. RSM was adopted to optimize the medium in order to obtain a good growth of the fungus as a prerequisite for enhanced sporulation. In the second step, the basal medium was supplemented with different trace elements which significantly affect sporulation (i.e. CuSO₄·5H₂O, FeCl₃·6H₂O, Co(NO₃)₂·6H₂O, and MnCl₂·4H₂O). Central composite design proved to be valuable in optimizing a chemically defined solid medium for spore production of B. trispora. The composition of the new solid medium to enhance spore production by B. trispora (ATCC 14271) is as follows (per liter): 7.5 g glucose, 3.2 g corn steep liquor, 1.7 g yeast extract, 4.1 g ammonium sulfate, 6 mg CuSO₄·5H₂O, 276 mg FeCl₃·6H₂O, 2 mg Co(NO₃)₂·6H₂O, and 20 g agar (pH 6.0). Practical validation of this optimum medium gave spore number of 1.2 × 10⁸ spores/dish which is 77% higher than that produced in Potato Dextrose Agar (PDA). In the case of B. trispora (ATCC 14272) the new solid substrate for enhanced sporulation consists of (per l) 6.4 g glucose, 3.3 g corn steep liquor, 1.4 g yeast extract, 4.3 g ammonium sulfate, 264 mg CuSO₄·5H₂O, 485 mg FeCl₃·6H₂O, 223 mg MnCl₂^.4H₂O, and 20 g agar (pH 6.0). Spore numbers of 2 × 10⁷ spores/dish were obtained on the new medium by B. trispora (ATCC 14272), which is 95% higher than that produced on PDA. The results corroborated the validity and the effectiveness of the models. The new media considerably improved sporulation of both strains of B. trispora compared to the production of spores on PDA, which is the medium usually used for sporulation of the fungus.     

The Amount of Phosphate Solubilization Depends on the Strain,C-Source,Organic Acids and Type of Phosphate

Although production of organic acids (OAs) is usually mentioned as the main mechanism of phosphate solubilization, the relationship between carbon sources (C-sources) and OAs produced during phosphate-solubilization by microorganisms is still poorly understood. We evaluated the influence of different C-sources on FePO₄·2H₂O and Ca₃(PO₄)₂ solubilization by bacteria and on the identity/quantity of the OAs produced. Our results showed that the amount of phosphate solubilization depends on the strain, C-source, OAs, and type of phosphate. Among the five strains under study isolated from cowpea nodules (Rhizobium tropici strain UFLA 03-08, Acinetobacter sp. strain UFLA 03-09, Paenibacillus kribbensis strain UFLA 03-10, P. kribbensis strain UFLA 03-106, and Paenibacillus sp. strain UFLA 03-116), three of them solubilized Ca₃(PO₄)₂ in all C-sources. The influence of C-sources on Ca₃(PO₄)₂-solubilization increased in the following order: cellulose?<?lactose?<?mannitol?<?glucose. A significant positive correlation between the amount of phosphorus solubilized from Ca₃(PO₄)₂ and the concentration of total OAs in the presence of glucose and mannitol was observed for these three strains. In the presence of glucose, the highest solubilization rates are associated with high concentrations of tartaric acid, and in the presence of mannitol, are associated with maleic acid. Only one strain produced OAs in the medium with lactose and Ca₃(PO₄)₂, but there was no OAs in the medium containing cellulose. Despite the production of OAs, albeit in small concentrations, in all the C-sources investigated, FePO₄·2H₂O-solubilization was not observed. Thus, a relationship among C-sources, OAs, and phosphate solubilization was not always verified.     

Isolation of hydrogen-producing bacteria from granular sludge of an upflow anaerobic sludge blanket reactor

H₂-producing bacteria were isolated from anaerobic granular sludge. Out of 72 colonies (36 grown under aerobic conditions and 36 under anaerobic conditions) arbitrarily chosen from the agar plate cultures of a suspended sludge, 34 colonies (15 under aerobic conditions and 19 under anaerobic conditions) produced H₂ under anaerobic conditions. Based on various biochemical tests and microscopic observations, they were classified into 13 groups and tentatively identified as follows: From aerobic isolates,Aeromonas spp. (7 strains),Pseudomonas spp. (3 strains), andVibrio spp. (5 strains); from anaerobic isolates,Actinomyces spp. (11 strains),Clostridium spp. (7 strains), andPorphyromonas sp. When glucose was used as the carbon substrate, all isolates showed a similar cell density and a H₂ production yield in the batch cultivations after 12h (2.24–2.74 OD at 600 nm and 1.02–1.22 mol H₂/mol glucose, respectively). The major fermentation by-products were ethanol and acetate for the aerobic isolates, and ethanol, acetate and propionate for the anaerobic isolates. This study demonstrated that several H₂ producers in an anaerobic granular sludge exist in large proportions and their performance in terms of H₂ production is quite similar.     

Hydrogen and Polyhydroxybutyrate Producing Abilities of <Emphasis Type="Italic">Bacillus</Emphasis> spp. From Glucose in Two Stage System

Metabolic activities of four Bacillus strains to transform glucose into hydrogen (H₂) and polyhydroxybutyrate (PHB) in two stages were investigated in this study. Under batch culture conditions, Bacillus thuringiensis EGU45 and Bacillus cereus EGU44 evolved 1.67–1.92 mol H₂/mol glucose, respectively during the initial 3 days of incubation at 37°C. In the next 2 days, the residual glucose solutions along with B. thuringiensis EGU45 shaken at 200 rpm was found to produce PHB yield of 11.3% of dry cell mass. This is the first report among the non-photosynthetic microbes, where the Bacillus spp.—B. thuringiensis and B. cereus strains have been shown to produce H₂ and PHB in same medium under different conditions.     

Differential medium for revelation of bacterial producer strains of L-asparaginases

A specific, fast, and easy method for revelation of active plate producers of L-asparaginase using differential medium on the basis of LB or M9 with 1.5% agar was developed. Each 100 ml of LB or M9 medium additionally contained 6–7 ml of glycerol, 4 g of L-asparagine, 0.2 g of CaCO₃, and diagnostic components: 3 ml of 0.2 M CuSO₄ · 5H₂O and 2.5 ml of 0.1 M K₃Fe(CN)₆, pH 7.6–7.8. The results were counted 12–20 or 24–48 h after strain growth at 37°C in corresponding mediums. Red color of colonies and colored zone around them showed the ability of the strain under study to destroy asparaginic complexes. The recommended method allows revealing bacterial strains producing L-asparaginase with specific activity of not less than 0.1–3.0 MU/mg of protein.     

Cultural Conditions and Some Properties of the Lipase of Humicola lanuginosa S-38

The cultural conditions for the production of thermostable lipase by a thermophilic fungus Humicola lanuginosa S-38 were investigated. The optimal cultural conditions to obtain the maximum yield of thermostable lipase with a 600-liter stainless steel fermentor were as follows: optimal medium- 2.0% soluble starch, 5.0% corn steep liquor, 0.2% K₂HPO₄, 0.1% MgSO₄·7H₂O, 0.5% CaCO₃, 0.5% soybean oil, 0.005% deforming agent (Adecanol LG-109); optimal fermentation conditions- temperature 45°C; rate of agitation 300 rpm; initial pH 7.0; rate of aeration 1/1 volume per volume of medium per minute. The optimal pH of the crude lipase preparation for the hydrolysis of the polyvinyl alcohol-emulsified olive oil was 8.0 and the optimal temperature was 60°C. It retained 100% of activity with the heat treatment at 60°C for 2 hr, but at 70°C for 20 min only 35% activity retained.     

Studies on Some New Thiazolidones as Potential Fungicides

The reaction conditions for the production of d-β-hydroxyphenylglycine (d-HPG) from dl-5-(β-hydroxyphenyl)hydantoin (dl-HPH) by cells of Pseudomonas sp. AJ-11220, and the cultural conditions for this bacterium for the formation of the d-HPG-producing enzyme involved by this bacterium were investigated. The optimal pH of this reaction was about 8.0 and the optimal temperature about 43°C. The d-HPG-producing enzyme was inducibly produced in Pseudomonas sp. AJ-11220 in proportion to the cell growth. Cells containing high activity were obtained when Pseudomonas sp. AJ-11220 was grown in a medium containing 20 g of glucose, 5g of (NH₄)₂SO₄,. 1 g of KH₂PO₄, 3g of K₂HPO₄, 0.5g of MgSO₄–7H₂O, 0.01 g of FeSO₄–7H₂O, 0.01 g of MnSO₄ -4H₂O, 10 g of yeast extract, 5g of dl-5-cyanoethylhydantoin and 20 g of CaCO₃ in a total volume of 1 liter (pH 7.0). Under the optimal conditions, 25 mg/ml of d-HPG was asymmetrically and directly produced from 30 mg/ml of dl-HPH with a molar yield of 92%. Various d-amino acids could also be effectively produced from the corresponding 5-substituted hydantoins.     

Optimizing Production of Extracellular Laccase from Grammothele Subargentea CLPS No. 436 Strain

The production of extracellular laccase by the Grammothele subargentea CLPS no. 436 strain in liquid cultures grown on a carbon-limited basal medium was significantly enhanced when culture conditions, including the addition of CuSO₄·5H₂O or veratryl alcohol, were consecutively optimized. A laccase activity as high as 1954.5 mU ml⁻¹ of liquid medium was obtained under optimum conditions, which corresponded to non-agitated cultures supplemented with 0.6 mM CuSO₄·5H₂O. Veratryl alcohol at 1 mM was less effective than CuSO₄·5H₂O for increasing laccase activity levels; the supplementation of veratryl alcohol resulted only in maximum levels of 44 mU ml⁻¹ in non-agitated cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Optimization of a Propionibacterium acidipropionici continuous culture utilizing sucrose

To produce propionic acid and vitamin B₁₂ from sucrose, the strain Propionibacterium acidipropionici NRRL B3569 was selected by screening a number of Propionibacterium strains. The nutrient composition and the fermentation conditions for this strain were optimized in continuous culture. The investigations show that within a concentration range of 30–170 g l^–1 of sucrose in the fermentation medium, no significant substrate inhibition occurred. For the production of propionic acid and vitamin B₁₂, concentrations of 1.5 mg FeSO₄·7H₂O g^–1 dry biomass, 0.75 mg cobalt ions g^–1 dry biomass, 0.3 mg 5,6-dimethylbenzimidazole g^–1 dry biomass, and 12 g yeast extract 1^–1 were necessary additions to the sources of nitrogen, phosphate, and magnesium ions. The extra addition of up to 2.8 g betaine g^–1 dry biomass significantly increases the production of vitamin B₁₂. In the optimization of the pH value, temperature, and aeration, it was established that the conditions for propionic acid production and vitamin B₁₂ production are different. Whereas the optimal production of propionic acid took place under completely anaerobic conditions with a pH value of 6.5 and a temperature of 37°C, optimal vitamin B₁₂ production required a temperature of 40°C and aerobic conditions (0.5 vvm aeration at 100 rpm) with a pH value of 6.5.     

Optimization of extracellular alkaline protease production from species of <Emphasis Type="Italic">Bacillus</Emphasis>

Thirty-five strains capable of secreting extracellular alkaline proteases were isolated from the soil and waste water near the milk processing plant, slaughterhouse. Strain APP1 with the highest-yield alkaline proteases was identified as Bacillus sp. The cultural conditions were optimized for maximum enzyme production. When the initial pH of the medium was 9.0, the culture maintained maximum proteolytic activity for 2,560 U ml⁻¹ at 50°C for 48 h under the optimized conditions containing (g⁻¹): soyabean meal, 15; wheat flour, 30; K₂HPO₄, 4; Na₂HPO₄, 1; MgSO₄·7H₂O, 0.1; Na₂CO₃, 6. The alkaline protease showed extreme stability toward SDS and oxidizing agents, which retained its activity above 73 and 110% on treatment for 72 h with 5% SDS and 5% H₂O₂, respectively.     

Stress Tolerance and Genetic Variability of Phosphate-solubilizing Fluorescent Pseudomonas from the Cold Deserts of the Trans-Himalayas

Nineteen efficient phosphate-solubilizing fluorescent Pseudomonas from the cold deserts of the trans-Himalayas were screened for stress tolerance against temperature, alkalinity, salinity, calcium salts, and desiccation. Phylogenetic analysis based on 16S rRNA gene sequencing placed these bacteria under three groups with fourteen strains in Group I including Pseudomonas trivialis and P. poae, two strains in Group II together with Pseudomonas kilonensis and P. corrugata, and three strains in Group III along with Pseudomonas jessenii and P. moraviensis. Genetic diversity assessed by ERIC and BOX-PCR revealed variability among strains belonging to the same phylogenetic groups. Cluster analysis based on the growth characteristics under regimes of different stress levels placed the strains into three distinct clusters displaying no correlation to their phylogenetic groups. Stress-tolerant strains differed in the level of decline in phosphate solubilization under increasing intensity of various stress parameters. The highest decrease occurred with 5% CaCO_3, followed by 2.5% CaCO₃, pH 11, 5% NaCl, temperature of 37°C, 40% PEG, 5% CaSO₄, 2.5% NaCl, 2.5% CaSO₄, pH 9 and temperature of 15°C. Two strains belonging to Phylogenetic Group I exhibited higher phosphate solubilization at lower temperature. The results revealed that stress-tolerance ability was not limited to any particular phylogenetic group. Knowledge about the genetic variants of phosphate-solubilizing fluorescent Pseudomonas with potential for tolerance to desiccation, alkalinity, temperature, and salinity could be useful in understanding their ecological role under stressful environments of low phosphate availability.     

Selectively Increased Production of Acidic Actinomycin Congener,B-1625 FA2β, on Combined Use of Sarcosine and Ferrous Sulfate

In addition to actinomycins D, X₂ and X_0β, Streptomyces antibioticus No. B-1625 produces minor acidic actinomycin congeners (FA-components). To increase the production of the FA-components, improvement of medium constituents was attempted for both chemically defined and complex media. Addition of trace metals, especially FeSO₄, increased FA-components production and, moreover, the addition of sarcosine was found to increase the production of a selected component, B-1625 FA_2β. Finally, a complex medium, consisting of starch 3.0, Polypepton 0.1, meat extract 0.1, corn steep liquor 3.0, NaCl 0.3, CaCO₃ 0.3, sarcosine 0.1 and FeSO₄ · 7H₂O 0.05%, was developed for the increased production of FA-components, in particular, the selected component of B-1625 FA_2β.