Fermentative Production of Chloramphenicol-analogs (Corynecins) from Sucrose

Corynecins (N-acyl derivatives of d-(?)-threo-p-nitrophenyl serinol), which were firstly discovered in the culture broth of Corynebacterium hydrocarboclastus KY 4339 grown on n-alkane were also produced when n-alkane was replaced by sucrose. The corynecins production in the sucrose-medium was significantly stimulated by the supplement of molasses. On the basis of the composition of ingredients in molasses, the preferable culture medium was designed for the production of corynecins from sucrose. This semi-synthetic medium is consisted of low concentration of phosphate, high concentration of potassium chloride, inositol, fructose and yeast extract in addition to ordinary mineral salts. By controlling the pH of the medium at the neutral range and keeping the aeration at a relatively high level, approximately 4 g of corynecins (as l-base) per liter of the medium were produced using a 5-liter jar fermentor.     

Production of Antibacterial Compounds Analogous to Chloramphenicol by a n-Paraffin-grown Bacterium

Corynebacterium sp. KY 4339, when grown on n-paraffin (a mixture of C–12 to C–14 fractions) as the sole carbon source, produced three kinds of antibacterial compounds which were tentatively named Corynecins. These compounds were isolated by the extraction from the culture broth with ethyl acetate and by the chromatographies on silicic acid and alumina columns. Each component demonstrated some similarity to chloramphenicol on thin-layer chromatogram. Although their biological activities were not so remarkably as that of chloramphenicol, the patterns of antibacterial spectra against gram-positive and gram-negative bacteria resembled to it.

For the production of corynecins, n-paraffin was a preferable carbon source. By controlling the pH of the medium in the neutral range and keeping the aeration at a high level during the fermentation, approximately 3 g of corynecins per liter of the medium were produced after 72-hr incubation.     

Production of Corynecins by Mutants Defective in Glycolipids Synthesis and Increased in Corynecin I

For the purpose of improving the productivity of corynecins (chloramphenicol analogs), the mutation study of the producer, Corynebacterium hydrocarboclastus, was concerned in this investigation with the isolation of glycolipids defective strains. The mutant KY 8834 possessed glycolipids less than one-third to one-tenth of the parent. Productivity of corynecins of the mutant was better than that of the parent, In addition, this character of the mutant was advantageous for the stirring fermentation, because aggregation of cells was remarkably diminished unlike the case of the parental strain.

Another mutant was isolated by mutagenesis of glycolipids defective mutant, KY 8834. The mutant, KY 8835, produced dominantly corynecin I (less toxic homolog to the producer strain). Thus, the productivity of corynecins was elevated to about two-fold of that of strain KY8834.     

Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes

Microorganisms which produced n-alkane ω,ω′-dicarboxylic acid (DC) from n-alkane were selected from natural sources. It was found that the best three producers thus obtained belonged to yeast. All of the stock cultures which are able to assimilate n-alkane and are belonged to genus Candida and Pichia were also found to produce DC from n-alkane.

Candida cloacae 310, a representative strain selected from natural source, was able to produce DCs having 5 to 16 carbon atoms from various n-alkanes. Among them, DCs with 5 to 9 carbon atoms were more heavily accumulated than those with more than 9, except those with the same number of carbon atoms as the substrates which were the main products from the substrates with less than 15 carbon atoms. It was also clearly demonstrated that DCs with odd carbons alone were produced from n-alkanes with odd carbons, while DCs with even carbons alone from n-alkanes with even carbons.

Then, cultural conditions of Candida cloacae 310 were studied for the production of DC-12 from n-dodecane (n-C₁₂) which showed the highest yield among the observed accumulation.

Under the optimum conditions, 2.28 g/liter of DC-12 was obtained together with 1.86 g/liter of DC-6 and 0.82 g/liter of DC-8 after 72 hr’ cultivation in a synthetic medium containing 100 ml of n-C₁₂ per liter.     

Effects of Corn Steep Liquor and Thiamine on l-Glutamic Acid Fermentation of Hydrocarbons: IV. Utilization of Hydrocarbons by Microorganisms

The effect of nutrients of natural source, such as corn steep liquor, peptone, and yeast extract, on the fermentative production of L-glutamic acid from hydrocarbons by a Corynebacterium was studied. Corn steep liquor and meat extract were found to be remarkably stimulatory to L-glutamic acid production; about 5 g per liter of L-glutamic acid were accumulated in a culture broth containing 3% n-paraffins, 0.01% corn steep liquor, and mineral salts. Among nutritional factors contained in corn steep liquor, biotin had very little effect on the accumulation of L-glutamic acid, but thiamine was highly stimulatory to L-glutamic acid production. The optimal concentration of thiamine for L-glutamic acid production was 3 to 5 μg per liter, and for cell growth, 50 μg per liter. L-Glutamic acid was accumulated in negligible quantity when the amount of thiamine in the culture broth was sufficient to support abundant growth of bacterial cells.     

Production of Inosine from Hydrocarbons by Corynebacterium petrophilum

Adenine-auxotrophic mutants were derived from Corynebacterium petrophilum SB 4082 by ultraviolet irradiation. The isolation of this auxotroph was carried out as follows; the adenine-auxotrophic mutants which had been derived by the UV irradiation were concentrated with penicillin. The auxotrophs were raised in concentration by recycling procedure and were separated by the ordinary method. The resultant adenine-auxotroph was cultured in the hydrocarbon medium. From its filtrate was obtained a fermentation product as crystals by means of ion-exchange resin and identified as inosine from absorption spectra and other properties.

The effects of cultural conditions on inosine production were investigated by the adenine auxotroph of Corynebacterium petrophilum SB 4082. That, the amount of adenine in the medium was very important on the inosine formation, was cleared. The addition of 10 mg of adenine and 0.5 g of yeast extract to 100 ml of medium was the best for the inosine formation. Ammonium chloride or ammonium sulphate was effective as nitrogen sources. As the carbon sources, n-C₁₀ to n-C₁₆ were utilized for the growth, but the hydrocarbons from n-C₁₂ to n-C₁₆ were the most suitable for the inosine formation. The inosine fermentation began at 24 hrs. after inoculation. The accumulation of inosine attained to the highest level after five days, the amount of which was 1.6 g per liter of the culture filtrate.     

Production of Corynecins by Chloramphenicol Resistant Mutants of Corynebacterium hydrocarboclastus

The isolation of chloramphenicol resistant strains from Corynebacterium hydrocarboclastus KY 4339 (rough type) was examined to seek a good source of corynecins (analogs of chloramphenicol). Various mutants resistant to chloramphenicol were isolated in the range from 50 to 1000 µg/ml by adaptation or induced mutagenesis by N-methyl-N′-nitro-N-nitro-soguanidine. Productivities of mutants related apparently to the degree of resistance from 50 to 500 µg/ml. Highly resistant mutants capable of growing in the presence of 1000 µg of chloramphenicol per ml showed decreased productivity which might be related to their lower growth rate in the fermentation medium.

Further attempts to derive resistant mutants to structural analogs of aromatic amino acids resulted in only a slight improvement of productivity, indicating that aromatic amino acids might play minor regulatory roles in corynecins synthesis.

The increase in productivity of corynecins by the best strain was about 4.5 fold of the parental strain.     

Anaerobic degradation of chlorophenol by an enrichment culture

Summary An anaerobic mixed culture from sewage sludge was enriched in a yeast extract and peptone-containing medium; it was able to degrade 2-cholorophenol completely to methane and CO₂. Degradation rates of 2-chlorophenol of up to 0.18 g/l per day were observed in suspended cultures without biomass retention and of 0.375 g/l per day in cultures immobilized on Liapor clay beads. Attempts to isolate the dechlorinating organism failed. The mixed culture was reduced to three morphologically distinctive microorganisms using a medium with limited amounts of yeast extract and peptone and n-butyrate as a co-substrate. Under these conditions the phenol-degrading bacterium was lost and phenol accumulated in the medium. No growth and no dehalogenation of 2-chlorophenol was obtained when yeast extract and peptone were omitted completely. Besides serving as a source of supplementary components, yeast extract and peptone were apparently required as the main source of carbon, wereas reducing equivalents for reductive dehalogenation were obtained by oxidation of n-butyrate. A spirochaete-like organism was presumably the dechlorinating bacterium. The mixed culture lost its dehalogenation capability if this organism was lost. n-Butyrate could be replaced by n-valerate, hexanoate, heptanoate, octanoate, pelargonic acid, n-decanoic acid or palmitate as co-substrates for dehalogenation of either 2-chlorophenol, 2-bromophenol or complete dechlorination of 2,6-dichlorophenol, whereas from 2,4-dichlorophenol only the substituent in the ortho-position could be eliminated.Dedicated to Professor O. Kandler on the occassion of his 70th birthdayOffprint requests to: J. Winter     

Optimization of <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation on sugarcane bagasse

Verticillium lecanii is an entomopathogen with high potential in biological control of pests. We developed a solid-state fermentation with sugarcane bagasse as carrier absorbing liquid medium to propagate V. lecanii spores. Using statistical experimental design, we optimized the medium composition for spore production. We first used one-factor-at-a-time design to identify corn flour and yeast extract as the best carbon and nitrogen sources for the spore production of V. lecanii. Then, we used two-level fractional factorial design to confirm corn flour, yeast extract, and KH₂PO₄ as important factors significantly affecting V. lecanii spore production. Finally, we optimized these selected variables using a central composite design and response surface method. The optimal medium composition was (grams per liter): corn flour 35.79, yeast 8.69, KH₂PO₄ 1.63, K₂HPO₄ 0.325, and MgSO₄ 0.325. Under optimal conditions, spore production reached 1.1 × 10¹⁰ spores/g dried carrier, much higher than that on wheat bran (1.7 × 10⁹ spores/g initial dry matter).     

Utilization of n-Paraffins and Vitamin B6 Production by Pichia guilliermondii Wickerham

The accumulation of vitamin B₆ by Pichia guilliermondii Wickerham NK–2 strain grown on hydrocarbon was investigated. Ammonium acetate was more effective than other nitrogen sources tested. Satisfactory utilization by the yeast strain was observed in n-alkanes of C₁₀–C₁₈, and n-pentadecane was the best for vitamin B₆ production. Vitamin B₆ was excreted in the cultural broth mainly in the form of pyridoxal, The maximal vitamin B₆ production was approximately 25 mg per liter of the culture broth.     

Thermoanaerobacter ethanolicus Growth and Product Yield from Elevated Levels of Xylose or Glucose in Continuous Cultures

The performance of Thermoanaerobacter ethanolicus was evaluated in continuous culture with media containing concentrations of xylose (8 to 20 g/liter) greater than those previously reported. The ethanol yield declined from to 0.42 to 0.29 g of ethanol per g of xylose consumed when input xylose was increased from 4 to 20 g/liter. Yields of both total C₂ and C₃ products from consumed xylose and of cell biomass from ATP produced declined as the input xylose concentration was increased, which was not the case when glucose was the substrate. This suggested that yeast extract functioned as a significant energy and carbon source for cells in fermentations of xylose but not of glucose. The feasibility of this interpretation was confirmed by (i) the calculation of the products theoretically obtainable from yeast extract and (ii) the observation of significant quantities of fermentation products in inoculated sugar-free media. Markedly different patterns of metabolism for the two sugar substrates were also evidenced by the cell yield for glucose being twice that of xylose at elevated sugar concentrations. It was noted that caution must be exerted when results obtained at low xylose concentrations are extrapolated to predict those which can be obtained at higher concentrations.     

Medium-Scale Production of Citrinin by Penicillium citrinum in a Semisynthetic Medium

A convenient method is described for the production of up to 1.75 g of citrinin per liter by Penicillium citrinum growing in stationary culture in a 5-gallon (18.925 liters) carboy containing 4 liters of 4% sucrose and 2% yeast extract medium.     

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and its nutritive analysis

After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0°C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C_16:0 (19.0%), C_18:0 (46.3%), and C_18:1 (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production.     

The Structures of Corynecins; Chloramphenicol Analogues Produced by a n-Paraffin-grown Bacterium

Corynebacterium sp. produced antibacterial substances (corynecins) when a n-paraffin mixture was used as the sole carbon sourse. Structures of the substances were determined as acyl derivatives of d-(?)-threo-l-p-nitrophenyl-2-amino-l,3-propanediol (free base of chloramphenicol).     

Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes

Strain M-l which was derived from Candida cloacae 310 as a mutant unable to assimilate dicarboxylic acid (DC) produced large amount of DCs from n-alkanes, as expected. It produced DCs with the same number of carbon atoms as those of n-alkanes used (9 to 18 carbon atoms). Among DCs produced, n-tetradecane ω,φ′-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) was most abundantly accumulated and the highest level of DC-16, i.e., 29.3g/liter was obtained by resting cells.

On the other hand, since the growth rate of strain M-l on n-alkane markedly decreased in comparison with that of the parent strain, other carbon source which supported the growth of strain M-l was necessary for the production of DC from n-alkane by growing cells. When acetic acid was used as carbon source for the growth in DC-16 production from n-C₁₆, the highest level of DC-16, i.e., 21.8 g/liter was obtained ofter 3 days' cultivation.     

Optimization of culture conditions for the production of rifamycin oxidase by Curvularia lunata

Maximum activity (8.9 IU/ml) of rifamycin oxidase in Curvularia lunata, grown in shake-flask culture at 28°C and pH 6.5, was after 96 h. Nearly all the glucose was used in 72 h. An initial culture pH of 6.5 and 28°C were optimum for the growth and enzyme production. Among various carbon and organic nitrogen sources, carboxymethylcellulose and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when yeast extract was also added to the medium. The optimum medium for the production of rifamycin oxidase contained 10 g each of yeast extract, peptone and carboxymethylcellulose/l and 0.04% (NH₄)₂SO₄.The author is with the Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Post Box 1304, Sector 39-A, Chandigarh 160 014, India     

Production of high-quality edible protein from Candida yeast grown in continuous culture

Candida utilis NRRL Y-900 was grown in aerobic continuous culture with cane molasses as the source of the growth-limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at D_max. On the average, the yeast biomass was 50–55% protein. At S_R > 2% sugar, the biomass productivity was limited by the oxygen supply. With O₂-supplemented aeration (at S_R = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.     

Tetradecane 1,14-Dicarboxylic Acid Production from n-Hexadecane by Candida cloacae

The better condition of cultivation for tetradecane 1,14-dicarboxylic acid (DC-16) production from n-hexadecane (n-C₁₆) by Candida cloacae MR-12 was investigated by using acetic acid as carbon source for the growth. In general, the condition suitable for the growth was also favorable for the production of DC-16. The change of pH during cultivation, the use of NaOH solution as pH controlling agent after pH-change and the addition of antifoam stimulated the production of DC-16.

Under the optimum conditions where the culture medium contained 15% (v/v) n-C₁₆, 1.4% (w/v) acetic acid, inorganic salts and growth factors, and pH was changed from 6.5 to 7.75 at 16 hr after the inoculation, the highest level of DC-16 production was attained after about 72 hr cultivation and the amount of the product accumulated was 61.5 g per liter of the medium.

When a mixture of various n-alkanes was used as starting material, DCs corresponding to the respective n-alkanes were produced as mixture.     

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.     

Malformin A1 as a Mammalian Toxicant from Aspergillus niger

Culture conditions for guanosine production were studied with Bacillus subtilis MG–1 that exclusively accumulated guanosine. Of components investigated, KH₂PO₄, KC1, Fe⁺⁺, Mn⁺⁺, NH₄NO₃ and sodium glutamate have played important roles for guanosine production. The optimal concentrations in the culture medium were 2.0 g, 0.9 g, 7.5 mg, 7.5 mg, 20. 4 g and 6.0 g per liter, respectively.

In particular, the extremely minor concentration of Mn⁺⁺ 0.01 ppm completely repressed guanosine production although the cells grew sufficiently. Amino acids mixture was necessary for cell growth, but not essential for guanosine production.

Under these conditions, MG–1 accumulated 15 g of guanosine per liter in a weight yield of 18.8% of consumed sugar. However, a large amount of acetoin was also found as a byproduct.