Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Effect of Yeast Extract and Vitamin B12 on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B₁₂, biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B₁₂ alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum

 Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55°C. The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling >100 kb, insuring that most potential restriction sequences have been exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5′ GATC 3′. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam⁺ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam^- phenotype. Received: 10 March 1995/Received revision: 4 September 1995/Accepted: 13 September 1995     

Growth Requirements of Gyrodinium cohnii*

SYNOPSIS. Gyrodinium cohnii, a marine heterotrophic dinoflagellate, was cultured at 35 C in a medium supplemented with yeast extract and finally in a defined medium. Vitamin B₁₂, not required at 30 C or lower, caused marked stimulation at 35 C used in combination with yeast extract or tryptophan + proline. Folic acid and possibly other vitamins may also be stimulatory at the elevated temperature. Ecological implications and applications to bioassays are discussed.     

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.     

Production of citric acid from methanol by a fluoroacetate-resistant mutant of Candida sp. Y-1

Summary Fermentative production of citric acid from methanol by an isolated yeast, Candida sp. Y-1, was investigated using a medium containing fluoroacetate, a potential inhibitor of aconitase. Culture conditions were optimized, and the results showed that efficient production of citric acid required several factors; (1) the optimum concentration of fluoroacetate, (2) an addition of yeast extract with corn steep liquor, (3) a low nitrogen source concentration, and (4) strictly aerobic conditions. We then isolated a fluoroacetate-resistant mutant strain MA92 with threefold higher citric acid productivity than the wild strain. This mutant strain had lower aconitase activity than the wild strain and produced 4.6 g/l citric acid from methanol after 4 days of culture. Offprint requests to: Y. Tani     

Isolation and Identification of a Microorganism which Produces Non Streptomyces Pepsin Inhibitor and N-Diazoacetyl-dl-norleucine Methylester Sensitive Acid Proteases

Non pepsin inhibitor (S–PI) and diazoacetyl-dl-norleucine methylester (DAN) sensitive acid proteases producing microorganism was isolated from farm soil of Osaka Prefecture.

The isolated strain was identified as Scytalidium lignicolum M–133. When it was aerobically grown on a medium consisting of glucose 5%, meat extract 1.5%, yeast extract 0.1%, KH₂PO₄ 0.2%, MgSO₄·7H₂O 0.05% at pH 3.5 and 25°C, the strain produced two acid proteases, A and B, in the culture broth.

The acid proteases A and B were not at all inactivated by S–PI and DAN. These acid proteases were expected to be a new type of acid protease from the viewpoint of the active site.     

Influence of organic and inorganic growth supplements on the aerobic biodegradation of chlorobenzoic acids

The effect of yeast extract and its less complex substituents on the rate of aerobic dechlorination of 2-chlorobenzoic acid (2-ClBzOH) and 2,5-dichlorobenzoic acid (2,5-Cl₂BzOH) by Pseudomonas sp. CPE2 strain, and of 3-chlorobenzoic acid (3-ClBzOH), 4-chlorobenzoic acid (4-ClBzOH) and 3,4-dichlorobenzoic acid (3,4-Cl₂BzOH) by Alcaligenes sp. CPE3 strain were investigated. Yeast extract at 50 mg/l increased the average dechlorination rate of 200 mg/l of 4-ClBzOH, 2,5-Cl₂BzOH, 3,4-Cl₂BzOH, 3-ClBzOH and 2-ClBzOH by about 75%, 70%, 55%, 7%, and 1%, respectively. However, in the presence of yeast extract the specific dechlorination activity of CPE2 and CPE3 cells (per unit biomass) was always lower than without yeast extract, although it increased significantly during the exponential growth phase. When a mixed vitamin solution or a mixed trace element solution was used instead of yeast extract the rate of 4-ClBzOH dechlorination increased by 30%–35%, whereas the rate of 2,5-Cl₂BzOH and 3,4-Cl₂BzOH dechlorination increased by only 2%–10%. The presence of vitamins or trace elements also resulted in a specific dechlorination activity that was generally higher than that observed for the same cells grown solely on chlorobenzoic acid. The results of this work indicate that yeast extract, a complex mixture of readily oxidizable carbon sources, vitamins, and trace elements, enhances the growth and the dechlorination activity of CPE2 and CPE3 cells, thus resulting in an overall increase in the rate of chlorobenzoic acid utilization and dechlorination.     

Isolation and characterization of a new CO-utilizing strain, Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans, isolated from a geothermal spring in Turkey

A novel anaerobic, thermophilic, Gram-positive, spore-forming, and sugar-fermenting bacterium (strain TLO) was isolated from a geothermal spring in Ayaş, Turkey. The cells were straight to curved rods, 0.4–0.6 μm in diameter and 3.5–10 μm in length. Spores were terminal and round. The temperature range for growth was 40–80°C, with an optimum at 70°C. The pH optimum was between 6.3 and 6.8. Strain TLO has the capability to ferment a wide variety of mono-, di-, and polysaccharides and proteinaceous substrates, producing mainly lactate, next to acetate, ethanol, alanine, H₂, and CO₂. Remarkably, the bacterium was able to grow in an atmosphere of up to 25% of CO as sole electron donor. CO oxidation was coupled to H₂ and CO₂ formation. The G + C content of the genomic DNA was 35.1 mol%. Based on 16S rRNA gene sequence analysis and the DNA–DNA hybridization data, this bacterium is most closely related to Thermoanaerobacter thermohydrosulfuricus and Thermoanaerobacter siderophilus (99% similarity for both). However, strain TLO differs from Thermoanaerobacter thermohydrosulfuricus in important aspects, such as CO-utilization and lipid composition. These differences led us to propose that strain TLO represents a subspecies of Thermoanaerobacter thermohydrosulfuricus, and we therefore name it Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans.     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.     

Sinorhizobium meliloti Cells Require Biotin and either Cobalt or Methionine for Growth

Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific. All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B₁₂-dependent homocysteine methyltransferase for methionine biosynthesis.     

Chemically Defined Minimal Medium for Growth of the Anaerobic Cellulolytic Thermophile Clostridium thermocellum

A minimal chemically defined medium has been developed for Clostridium thermocellum. The growth factors required are biotin, pyridoxamine, vitamin B₁₂, and p-aminobenzoic acid.     

Medium optimization for glucoamylase production by a yeast,Pichia subpelliculosa ABWF-64, in submerged cultivation

An amylolytic yeast strain Pichia subpelliculosawas shown to produce glucoamylase in submerged cultivation. The yeast strain produced the enzyme optimally at 30 °C and pH 5.6 in shake flasks agitated at 200 rev min^–1 in the optimized glucoamylase production medium containing 1% starch, 0.2% yeast extract, 0.4% K₂HPO₄, 0.035% NaCl and 0.1% MgCl₂. Maximum enzyme production was attained during early growth of 11 h in shake flasks, and 6 h in a laboratory fermenter. By optimizing media components and cultivation parameters, a 15-fold increase in glucoamylase secretion was achieved.     

Thermococcus alcaliphilus sp. nov., a new hyperthermophilic archaeum growing on polysulfide at alkaline pH

A novel coccoid-shaped, hyperthermophilic, heterotrophic member of the archaea was isolated from a shallow marine hydrothermal system at Vulcano Island, Italy. The isolate grew between 56 and 90° C with an optimum around 85° C. The pH range for growth was 6.5 to 10.5, with an optimum around 9.0. Polysulfide and elemental sulfur were reduced to H₂S. Sulfur stimulated the growth rate. The isolate fermented yeast extract, peptone, meat extract, tryptone, and casein. Isovalerate, isobutyrate, propionate, acetate, CO₂, NH₃, and H₂S (in the presence of S°) were detected as end products. Growth was not inhibited by H₂. Based on DNA-DNA hybridization and 16S rRNA partial sequences, the new isolate represents a new species of Thermococcus, which we named Thermococcus alcaliphilus. The type strain is isolate AEDII12 (DSM 10322) Received: 7 July 1995 / Accepted: 25 August 1995     

Modulation of the Purine Pathway for Riboflavin Production in Flavinogenic Recombinant Strain of the Yeast Candida famata

Riboflavin (vitamin B₂) is an indispensable nutrient for humans and animals, since it is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in variety of metabolic reactions. Riboflavin is produced on commercial scale and is used for feed and food fortification purposes, and in medicine. Until recently, the mutant strains of the flavinogenic yeast Candida famata were used in industry for riboflavin production. Guanosine triphosphate is the immediate precursor of riboflavin synthesis. Therefore, the activation of metabolic flux toward purine nucleotide biosynthesis is a promising approach to improve riboflavin production. The phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase are the rate limiting enzymes in purine biosynthesis. Corresponding genes PRS3 and ADE4 from yeast Debaryomyces hansenii are modified to avoid feedback inhibition and cooverexpressed on the background of a previously constructed riboflavin overproducing strain of C. famata. Constructed strain accumulates twofold more riboflavin when compared to the parental strain.     

Nutritional requirements ofPrevotella sp. Isolated from the rumen of the goat

The nutritional requirements forPrevotella sp. 4PCCNB2 isolated from the rumen of a native goat in Korea and those of the ATCC 19189 strain isolated from the bovine rumen were investigated. The two strains grew well with ammonium sulfate as the sole added nitrogen source. However, neither a complex of amino acids nor casein hydrolysate effectively replaced ammonium sulfate. Biotin,p-aminobenzoic acid, and vitamin B₁₂ were essential to culture the ATCC 19189 strain. Unlike the ATCC 19189 strain, however, B₁₂ was only stimulatory for the growth of the 4PCCNB2 strain. The 4PCCNB2 strain grew well in the basal medium without an individual acid such as acetic acid or valeric acid. In contrast, either acetic or valeric acid was absolutely required for the growth of the ATCC 19189 strain.     

Production of isomeric 9,10,13 (9,12,13)-trihydroxy-11E (10E)-octadecenoic acid from linoleic acid by Pseudomonas aeruginosa PR3

Trihydroxy unsaturated fatty acids with 18 carbons have been reported as plant self-defense substances. Their production in nature is rare and is found mainly in plant systems. Previously, we reported that a new bacterial isolate, Pseudomonas aeruginosa PR3, converted oleic acid and ricinoleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid and 7,10,12-trihydroxy-8(E)-octadecenoic acid, respectively. Here we report that strain PR3 converted linoleic acid to two compounds: 9,10,13-trihydroxy-11(E)-octadecenoic acid (9,10,13-THOD) and 9,12,13-trihydroxy-10(E)-octadecenoic acid (9,12,13-THOD). Stereochemical analyses showed the presence of 16 different diastereomers — the maximum number possible. The optimum reaction temperature and pH for THOD production were 30°C and 7.0, respectively. The optimum linoleic acid concentration was 10 mg/ml. The most effective single carbon and nitrogen sources were glucose and sodium glutamate, respectively. However, when a mixture of yeast extract (0.05%), (NH₄)₂HPO₄ (0.2%), and NH₄NO₃ (0.1%) was used as the nitrogen source, THOD production was higher by 8.3% than when sodium glutamate was the nitrogen source. Maximum production of total THOD with 44% conversion of substrate was achieved at 72 h of incubation, after which THOD production plateaued up to 240 h. THOD production and cell growth increased in parallel with glucose concentration up to 0.3%, after which cell growth reached its maximum and THOD production did not increase. These results suggested that THODs were not metabolized by strain PR3. This is the first report of microbial production of 9,10,13- and 9,12,13-THOD from linoleic acid. Journal of Industrial Microbiology & Biotechnology (2000) 25, 109–115. Received 18 March 2000/ Accepted in revised form 09 June 2000     

Effect of Yeast Extract on Growth and Metabolism of H2-Utilizing Acetogenic Bacteria from the Human Colon

The aim of this work was to determine the effect of yeast extract and of its vitamin contents on autotrophic and heterotrophic growth and metabolism of four acetogenic bacteria from the human colon. Yeast extract exerted a stimulatory effect on autotrophic growth of the colonic acetogens, but concentration of this compound above 1–2 g. L⁻¹ in the medium did not enhance utilization of H₂/CO₂. Vitamins provided by yeast extract were shown to be essential cofactors of the reductive pathway of acetate synthesis except for one Clostridium strain. Yeast extract was also necessary to maintain heterotrophic growth and acetate synthesis from glucose in acetogenic species, except in the Streptococcus strain. In the absence of yeast extract, vitamins could efficiently restore glucose fermentation via acetate. The reductive and oxidative pathways of acetate synthesis might, therefore, depend on vitamin cofactors supplied by yeast extract in most of the human acetogenic bacteria. Non-vitaminic factors appeared also to be involved in the metabolism of some of these acetogenic species. Received: 6 March 1998 / Accepted: 3 April 1998     

Improved Microbial Production of Colominic Acid,a Homopolymer of N-Acetylneuraminic Acid

A culture medium has been devised for producing colominic acid in improved yields. Major improvements were obtained by using sorbitol as a source of carbon, by adding phosphate in high concentrations, and by supplementing a limited amount of yeast extract. E. coli O 16: Kl: HNM produced approximately 3000 µg/ml of colominic acid on cultivation at 37°C for 46 hr with a liquid medium consisting of sorbitol (2.0%), (NH₄)₂SO₄ (0.5%), K₂HPO₄ (1.4%), MgSO₄·7H₂O (0.05%), and yeast extract (0.05%).

Isolation and purification by deproteinization with ammonium sulfate, precipitation with ethanol, and by column chromatography on anion exchange resins resulted in a pure colominic acid preparation devoid of internal ester linkages.

In producing colominic acid, strains forming S-type colonies were more active than those forming R-type colonies.     

Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.