Development of balanced medium composition for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3

Aim:  To develop optimum fermentation environment for enhanced rifamycin B production by isolated Amycolatopsis sp. RSP-3.
Methods and Results:  The impact of different fermentation parameters on rifamycin B production by isolated Amycolatopsis sp. RSP-3 was investigated using Taguchi methodology. Controlling fermentation factors were selected based on one variable at a time methodology. The isolated strain revealed more than 25% higher production compared to literature reports. Five different nutritional components (soyabean meal, glucose, potassium nitrate, calcium carbonate and barbital) and inoculum concentration showed impact on rifamycin B production at individual and interactive level. At optimized environment, 65% contribution was observed from selected fermentation parameters.
Conclusions:  Soyabean meal and calcium carbonate were the most significant factors among the selected factors followed by barbital and potassium nitrate. Glucose, however, showed the least significance on rifamycin B production with this strain. A maximum of 5·12 g l⁻¹ rifamycin B production was achieved with optimized medium containing (g l⁻¹) soyabean meal, 27; glucose, 100; potassium nitrate, 4; calcium carbonate, 3 and barbital, 1·2.
Significance and Impact of the Study:  The present study signifies identification of balanced medium component concentrations for improved rifamycin B production by isolated Amycolatopsis sp. RSP-3. This strain requires organic and inorganic nitrogen sources for effective product yield. Yet at individual level, organic nitrogen source has c. nine-fold higher influence compared to inorganic one.     

Effects of kinetin and nitrogen on growth rates,pigment and protein contents in wild and phycoerythrin-deficient strains of <Emphasis Type="Italic">Hypnea musciformis</Emphasis> (Rhodophyta)

Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. is the main source for carrageenan production in Brazil and strains with selected characteristics could improve the production of raw material. The effects of kinetin on growth rates, morphology, protein content, and concentrations of pigments (chlorophyll a, phycoerythrin, phycocyanin, and allophycocyanin) were assessed in the wild strain (brown phenotype) and in the phycoerythrin-deficient strain (green phenotype) of H. musciformis. Concentrations of kinetin ranging from 0 to 50 μM were tested in ASP 12-NTA synthetic medium with 10 μM nitrate (N-limited) and 100 μM nitrate (N-saturated). In N-limited condition, kinetin stimulated growth rates of the phycoerythrin-deficient strain and formation of lateral branches in both colour strains. Kinetin stimulated protein biosynthesis in both strains. However, differences between both nitrogen conditions were significant only in the phycoerythrin-deficient strain. In the wild strain, effects of kinetin on concentrations of phycobiliproteins were not significant in both nitrogen conditions, except for chlorophyll content. However, the phycoerythrin-deficient strain showed an opposite response, and kinetin stimulated the phycobiliprotein biosynthesis, with the highest concentrations of phycoerythrin in N-saturated medium, while the highest concentrations of allophycocyanin and phycocyanin were observed in N-limited medium. These results indicate that the effects of kinetin on growth, morphology, protein and phycobiliprotein contents are influenced by nitrogen availability, and the main nitrogen storage pools in phycoerythrin-deficient strain of H. musciformis submitted to N-limited conditions were phycocyanin and allophycocianin, the biosynthesis of which was enhanced by kinetin.     

Mannitol production by heterofermentative <Emphasis Type="BoldItalic">Lactobacillus reuteri</Emphasis> CRL 1101 and <Emphasis Type="BoldItalic">Lactobacillus fermentum</Emphasis> CRL 573 in free and controlled pH batch fermentations

Certain lactic acid bacteria, especially heterofermentative strains, are capable to produce mannitol under adequate culture conditions. In this study, mannitol production by Lactobacillus reuteri CRL 1101 and Lactobacillus fermentum CRL 573 in modified MRS medium containing a mixture of fructose and glucose in a 6.5:1.0 ratio was investigated during batch fermentations with free pH and constant pH 6.0 and 5.0. Mannitol production and yields were higher under constant pH conditions compared with fermentations with free pH, the increase being more pronounced in the case of the L. fermentum strain. Maximum mannitol production and yields from fructose for L. reuteri CRL 1101 (122 mM and 75.7 mol%, respectively) and L. fermentum CRL 573 (312 mM and 93.5 mol%, respectively) were found at pH 5.0. Interestingly, depending on the pH conditions, fructose was used only as an alternative external electron acceptor or as both electron acceptor and energy source in the case of the L. reuteri strain. In contrast, L. fermentum CRL 573 used fructose both as electron acceptor and carbon source simultaneously, independently of the pH value, which strongly affected mannitol production by this strain. Studies on the metabolism of these relevant mannitol-producing lactobacilli provide important knowledge to either produce mannitol to be used as food additive or to produce it in situ during fermented food production.     

Glycosylation and production characteristics of epothilones in alkali-tolerant <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> strain So0157-2

3-O-α-D-ribofuranosyl epothilone A (epothiloneoside A) is a major component of glycosylated epothilones in Sorangium cellulosum strain So0157-2. The production and glycosylation ratios of epothiloneoside A in both solid and liquid culture conditions with various pH values and carbon sources were studied. The results showed that glycosylation occurs whenever epothilones are produced, regardless of changes in pH values, production time curves, and different carbon sources. We suggest that glycosylation is a stable process, paralleling the biosynthesis of epothilones in the So0157-2 strain.     

一株高产洛伐他汀红曲霉的筛选与液态发酵条件优化

【背景】洛伐他汀(lovastatin)是红曲霉的次生代谢产物,是重要的临床用降血脂药物。在液态发酵条件下,红曲霉的洛伐他汀产量较低,难以满足工业化生产的要求。【目的】筛选获得一株高产洛伐他汀的红曲霉株,并通过优化液态发酵条件提高洛伐他汀的产量。【方法】从红曲米中筛选获得一株高产洛伐他汀的红曲霉株,依据形态学特征、生理生化特性及18S rRNA基因序列分析对分离菌株进行鉴定;通过响应面法对其产洛伐他汀的液态发酵条件进行优化。【结果】获得一株产洛伐他汀的紫红曲霉(Monascus purpureus M4),该菌在甘油57.80g/L、酵母浸粉5.52 g/L、接种量为6.90%条件下,洛伐他汀产量(173.60 mg/L)较优化前提高了4.8倍。【结论】菌株M4产洛伐他汀最优液态发酵条件的建立,为洛伐他汀的大规模生产及该菌株的工业化应用提供了技术支撑。     

The donor side of photosystem II is impaired in a Cd-tolerant mutant strain of the unicellular green algaChlamydomonas reinhardtii

Growth of a cadmium-tolerant mutant strain of the unicellular green alga Chlamydomonas reinhardtii was found to be impaired under photoautrotrophic, but not under mixotrophic conditions. As compared to wild-type cells, oxygen evolution by the photoautotrophically grown mutant was considerably decreased and higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The capability for oxygen production was decreased in Chlamydomonas reinhardtii cells when grown in the presence of acetate without aeration. Wild-type cells grown under these conditions showed a rather low but significant oxygen evolution immediately after transfer to photoautotrophic conditions. This residual oxygen production was completely suppressed in the presence of acetate, obviously due to acetate inhibition of the water-splitting complex. In the case of our cadmium-tolerant mutant strain, however, residual oxygen production was measured even in the presence of acetate. After removal of acetate, oxygen evolution by the cadmium-tolerant mutant strain was increased to higher rates than measured for wild-type cells, but considerably higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The conclusion that the donor side of photosystem II is affected in our cadmium-tolerant mutant strain was further corroborated by a stronger decrease of the fluorescence level caused by hydroxylamine.     

Ethanol production from AFEX pretreated corn fiber by recombinant bacteria

Summary Fermentation of an enzymatic hydrolyzate of ammonia fiber explosion (AFEX) pretreated corn fiber (containing a mixture of different sugars including glucose, xylose, arabinose, and galactose) by genetically-engineered Escherichia coli strain SL40 and KO11 and Klebsiella oxytoca strain P2 was investigated under pH-controlled conditions. Both E. coli strains (SL40 and KO11) efficiently utilized most of the sugars contained in the hydrolyzate and produced a maximum of 26.6 and 27.1 g/l ethanol, respectively, equivalent to 90 and 92% of the theoretical yield. Very little difference was observed in cell growth and ethanol production between fermentations of the enzymatic hydrolyzate and mixtures of pure sugars, simulating the hydrolyzate. These results confirm the fermentability of the AFEX-treated corn fiber hydrolyzate by ethanologenic E. coli. K.oxytoca strain P2, on the other hand, showed comparatively poor growth and ethanol production (maximum 20 g/l) from both enzymatic hydrolyzate and simulated sugar mixtures under the same fermentation conditions.     

Concomitant loss of respiratory chain NADH: ubiquinone reductase (complex I) and citric acid accumulation in Aspergillus niger

Summary Growth, citric acid production and enzymatic activity of the mitochondrial respiratory enzymes of a wild-type and a citric-acid-producing mutant of Aspergillus niger have been compared during fermentation under citric-acid-accumulating and non-accumulating conditions. Under non-accumulating conditions, both strains showed standard growth and no citric acid production. The mutant strain was characterized by delayed onset of growth and lowered cell yield. Under citric-acid-accumulating conditions the wild-type strain exhibited decelerated growth and a maximal citric acid concentration of 12 g l^–1. Reduced, but continuing growth and citric acid production of 32 g l^–1 was observed for the mutant strain. In general, the mutant strain exhibited reduced activity for the proton-pumping respiratory complexes and enhanced activity for the alternative respiratory enzymes. In contrast to the stable activity of complex I in the wild-type strain, this complex was selectively lost in the mutant strain at the onset of citric acid production, while the alternative NADH dehydrogenases were kept at enhanced and constant activity. A possible causal connection between the loss of complex I and citric acid accumulation is discussed. Offsprint requests to: J. Wallrath     

Phenol degradation by immobilized cold-adapted yeast strains of Cryptococcus terreus and Rhodotorula creatinivora

Three strains were isolated from hydrocarbon-polluted alpine habitats and were representatives of Cryptococcus terreus (strain PB4) and Rhodotorula creatinivora (strains PB7, PB12). All three strains synthesized and accumulated glycogen (both acid- and alkali-soluble) and trehalose during growth in complex medium containing glucose as carbon source and in minimal salt medium (MSM) with phenol as sole carbon and energy source. C. terreus strain PB4 showed a lower total accumulation level of storage compounds and a lower extracellular polysaccharides (EPS) production than the two R. creatinivora strains, PB7 and PB12. Biofilm formation and phenol degradation by yeast strains attached to solid carriers of zeolite or filter sand were studied at 10°C. Phenol degradation by immobilized yeast strains was always higher on zeolite compared with filter sand under normal osmotic growth conditions. The transfer of cells immobilized on both solid supports to a high osmotic environment decreased phenol degradation activity by all strains. However, both R. creatinivora PB7 and PB12 strains maintained higher ability to degrade phenol compared with C. terreus strain PB4, which almost completely lost its phenol degradation activity. Moreover, R. creatinivora strain PB7 showed the highest ability to form biofilm on both carriers under high osmotic conditions of cultivation.     

Degradation of Benzene,Toluene and Xylene by Pseudomonas aeruginosa Engineered with the Vitreoscilla Hemoglobin Gene

This study concerns the potential use of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene for the degradation of important harmful aromatic compounds such as benzene, toluene, and xylene (BTX). The use of these compounds by both strains was determined as the production of cell mass (viable cell number) in a minimal medium containing any one of the BTX compounds as the sole carbon and energy source. Furthermore, the BTX degradation capability of both strains was monitored by measuring the production of 3‐methylcatechol, a common intermediate. For the cells of the logarithmic phase, which were grown at high aeration/high agitation or low aeration/low agitation, the engineered strain showed a better growth rate than the host strain. With the benzene in the medium, the recombinant strain exhibited a higher (up to 4‐fold) cell density than the parental wild‐type strain at this phase. In contrast, regarding the cells of the late stationary phase under high aeration/high agitation conditions, the host strain had generally higher viable cell numbers than the recombinant strain. At this phase this difference was, however, less significant under the conditions of low aeration/low agitation. Similarly, in toluene containing medium (at high aeration/high agitation) the recombinant strain showed a higher cell density which was from a 15‐fold to almost one order of magnitude greater than its parental strain during the logarithmic phase where the cell density of P. aeruginosa remained nearly constant. Contrary to the results with benzene and toluene, both strains exhibited similar growth characteristics when they were grown in the presence of xylene. The positive effect of the oxygen uptake by the recombinant system on the BTX metabolizing activity was also apparent in a high accumulation of 3‐methylcatechol in the cultures of the recombinant strain. At certain points of incubation, the hemoglobin expressing strain showed a significantly (p < 0.05) higher 3‐methylcatechol accumulation than the host strain. These results demonstrated the possible potential of the Vitreoscilla hemoglobin as an efficient oxygen uptake system for the bioremediation of some compounds of environmental concern.     

Fate in water of a recombinantEscherichia coli K-12 strain used in the commercial production of bovine somatotropin

Summary The fate in water ofEscherichia coli K-12 strain LBB269, both plasmid-free and carrying the recombinant plasmid pBGH1, was studied.E. coli K-12 strain LBB269 (pBGH1) is a nalidixic acid resistant derivative of W3110G (pBGH1), the microorganism used by Monsanto Company for the commercial production of bovine somatotropin. Water samples were obtained from the Missouri River and from the Monsanto Life Sciences Research Center aqueous waste basin. Strains LBB269 and LBB269 (pBGH1) were grown in fermentation vessel under bovine somatotropin (BST) production conditions, and inoculated into the water samples. The inoculated water samples were incubated, at 26°C, and the number of viableE. coli cells was determined as a function of time. In sterile water from both sources, the two strains remained, at a constant level for at least 28 days; LBB269 (pBGH1) remained at a constant level in sterile water for at least 300 days. In non-sterile water from both sources, the two strains declined from an initial concentration of about 3.0×10⁶ cells per ml to less than 10 cells per ml in 147 h. The study conditions did not adversely affect the populations of indigenous microorganisms. The selective loss of strains LBB269 and LBB269 (pBGH1) demonstrates that theseE. coli strains do not survive in environmental sources of water. In addition, it was observed that the presence of pBGH1 had essentially no effect on the disappearance of strain LBB269 from either source of water.     

Optimization of physicochemical parameters for the enhancement of carbonyl reductase production by Candida viswanathii

Culture conditions have been optimized for a newly isolated yeast strain Candida viswanathii PBR2 which is capable of reducing a wide variety of aryl ketones with high stereospecificity. Studies on the culture conditions and catalytic performance of this microorganism showed that the carbonyl reductase occurs constitutively in the cells and its production is enhanced by feeding with acetophenone (2 mM) during the early period of cultivation. Mannitol (1%, wv⁻¹) was found to be beneficial both for growth and enzyme production. Supplementation of the media with yeast extract (1.0%, wv⁻¹) and Ca²⁺ (4 mM) enhanced the enzyme production. The optimal temperature and pH for the growth and enzyme production were 25 °C and 9.0, respectively. Excellent conversions along with almost absolute enantioselectivity were observed when the resting cells of this yeast strain were exploited to carry out the stereoselective reduction of a number of aryl ketones.     

Survival of the Aerobic Denitrifier Pseudomonas stutzeri Strain TR2 during Co-Culture with Activated Sludge under Denitrifying Conditions

The aerobic denitrifier Pseudomonas stutzeri TR2 (strain TR2) has the potential to reduce nitrous oxide emissions during the wastewater treatment process. In this application, it is important to find the best competitive survival conditions for strain TR2 in complex ecosystems. To that end, we examined co-cultures of strain TR2 with activated sludge via five passage cultures in a medium derived from treated piggery wastewater that contained a high concentration of ammonium. The results are as follows: (i) The medium supported the proliferation of strain TR2 (P. stutzeri strains) under denitrifying conditions. (ii) Nitrite was a better denitrification substrate than nitrate for TR2 survival. (iii) Strain TR2 also demonstrated strong survival even under aerobic conditions. This suggests that strain TR2 is effectively augmented to the wastewater treatment process, aiding in ammonium-nitrogen removal and reducing nitrous oxide production with a partial nitrification technique in which nitrite accumulates.     

Characterization of Growth and Metabolism of Commercial Strains of Propionic Acid Bacteria by Pressure Measurement

Dairy propionibacteria are essential starters for Emmental cheese manufacture. The behavior of three commercial strains of Propionibacterium freudenreichii subsp. shermanii (P.f. 1, P.f. 2 and P.f 3) were studied in a liquid medium under air and N₂ atmosphere using an on‐line pressure measurement technique. Growth kinetics and metabolite production were characterized under conditions usually reported as “optimal conditions” (pH 6.5, NaCl 0 %, temperature 30 °C) and also evaluated under “stressful conditions” (pH 5.2, NaCl 2 %, temperature 20 °C) simulating the cheese ripening conditions. In both cases, the effects of oxygen on growth were strain‐dependent. Under “stressful conditions”, two of the three strains were inhibited by oxygen under conditions of air atmosphere, while all three strains grew under conditions of N₂ atmosphere. In the latter case, the duration of the lag phase and the maximum rate of pressure variation were significantly different, however, no significant differences were found between the strains with regard to the total fermentation time. Under “optimal conditions” metabolite production was strain‐dependent. In an air atmosphere, all strains produced more acetate and CO₂ and less propionate than in a nitrogen atmosphere.     

Characterization of a New <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strain NJ-15 as a Potential Biocontrol Agent

Phylogenetic characterization of soil isolate NJ-15, based on sequence homology of a partial 746-bp fragment of 16SrDNA amplicon, with the ribosomal database sequences (http://www.msu.edu/RDP/cgis/phylip.cgi), validated the strain as Pseudomonas aeruginosa. The strain NJ-15 produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. Besides, the strain also exhibited significant production of both the siderophore and hydrogen cyanide (HCN) on chrome azurol S and King's B media, respectively. The data revealed lower HCN production under iron-limiting conditions vis-à-vis higher HCN release with iron stimulation. Significant growth inhibition of phytopathogenic fungi occurred in the order as Fusarium oxysporum > Trichoderma herizum > Alternaria alternata > Macrophomina phasiolina upon incubation with strain NJ-15 cells. Thus, the secondary metabolites producing new Pseudomonas aeruginosa strain NJ-15 exhibited innate potential of plant growth promotion and biocontrol activities in vitro. Received: 30 April 2002 / Accepted: 5 July 2002     

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     

Improving the production of monensin byStreptomyces cinnamonensis

The production of monensin byStreptomyces cinnamonensis was increased by genetic improvement of the strain and by modification of cultivation conditions. The selection of a suitable strain and optimization of the fermantation process (temperature, aeration, addition of esters of oleic acid) resulted in a 30 fold increase of the monensin production.     

Optimal culture conditions for keratinase production by a novel thermophilic Myceliophthora thermophila strain GZUIFR‐H49‐1

Aims: To investigate the effect of medium compositions and culture conditions on keratinase production by a novel thermophilic fungus Myceliophthora thermophila (Apinis) Oorschot strain GZUIFR‐H49‐1. Methods and Results: The thermophilic strain GZUIFR‐H49‐1 with keratinolytic ability was characterized and identified as a strain of M. thermophila on the basis of its morphological characters and molecular analysis of ITS1‐5.8S‐ITS2 rDNA sequence. Among the medium compositions tested, the soluble starch (SS), urea, sodium thiosulfate and CaCl₂ were the most effective C‐source, N‐source, S‐source and mineral ion, respectively, by employing the single‐factor experiment. The urea and pH value were the significant factors (P < 0·05) for the keratinase production in this experiment condition using Plackett–Burman factorial design. The conditions of keratinase production were further optimized by Box–Behnken design. Consequently, there was a 6·4‐fold increase (5100 U l^?1) in the keratinase activity than the initial value (800 U l^?1) by this optimal process. Conclusions: This study indicated that the optimization design proved a useful and powerful tool for the development of optimal medium compositions and culture conditions. Myceliophthora thermophila strain GZUIFR‐H49‐1 was a promising fungus strain for keratinase production. Significance and Impact of the Study: This study characterized a novel thermophilic M. thermophila strain GZUIFR‐H49‐1 with potential applications for keratinase production. These conditions of keratinase production obtained by means of optimization design will be accumulated as potential information for exploration and utilization to the new fungus isolate.     

Characterization of hydrogen production by engineered Escherichia coli strains using rich defined media

Fermentation conditions (e.g., pressure and medium) are well-documented to impact the yield of microbial products in bioreactors. In this study we used carefully controlled batch fermentations to characterize hydrogen production from engineered strains of Escherichia coli and developed a rapid method of inducing hydrogen production in previously aerobically grown cells by using a rich defined medium. Our results indicated that rich defined media activated hydrogen production from aerobic pre-cultures with no lag time and yielded more hydrogen and biomass than the commonly used minimal media. Under these conditions, deletion of both uptake hydrogenase 1 (ΔhyaAB) and hydrogenase 2 (ΔhybABC) was shown to increase hydrogen yield from glucose by 10% over the wildtype strain BW25113. However, the deletion of the repressor for the formate-hydrogen-lyase (FHL-1) complex (ΔhycA) did not further increase hydrogen production. Additional deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdBC) of the mixed-acid fermentation pathway increased hydrogen yield by 22 and 23%, respectively. Interestingly, combined elimination of ldhA and frdBC in the uptake and hycA null strain increased hydrogen yield from 1.37 to 1.82 mol/mol glucose, obtaining 91% of the theoretical maximum hydrogen yield. Our results indicated the advantage of using rich defined media for inducing hydrogen production. This study represents the first report of characterizing metabolically engineered E. coli strains in batch hydrogen fermentation using rich defined media under tightly controlled conditions.     

Effect of environmental conditions on sclerotia and cleistothecia production in aspergillus

Summary Literature pertaining to sclerotial Aspergilli has been reviewed in brief. Observations on the effect of certain environmental conditions viz. pH, light, temperature of incubation, oxygen-deficient conditions and various relative humidity values on sclerotia production byAspergillus niger van Tieghem, (two strains),A. flavus Link (two strains),A. sclerotiorum Hüber (one strain) andA. paradoxus Fennell &Raper (one strain) and on cleistothecia production byA. nidulans (Eidam)Wint. (one strain) have been presented. Optimum pH for sclerotia or cleistothecia production was 7.5. In other respects sclerotia and cleistothecia behaved similarly. In general, condition showing maximum sclerotia or cleistothecia production was the one that showed maximum vegetative growth. Certain strains of the same species reponded differently to the same condition. Light completely inhibited sclerotia formation in one strain ofA. flavus. InA. paradoxus, in general, conditions favouring sclerotia production were those that inhibited (or retarded) the formation of conidial heads and the yellow pigment in the medium. Oxygen-deficient conditions inhibited or retarded sclerotia or cleistothecia formation. Production of sclerotia and cleistothecia increased with an increase in relative humidity values. No definite correlation could be observed between extent of sporulation and sclerotia or cleistothecia production except in case of relative humidity. Parallelism in the behaviour of sclerotia and cleistothecia production inAspergillus lends further support in favour of the hypothesis that in this genus sclerotia are sterile stromata.