E. G. Dedyukhina

Arachidonic acid production by Mortierella alpina with growth-coupled lipid synthesis

Abstract The fungus Mortierella alpina LPM 301, a producer of arachidonic acid (ARA), was found to possess a unique property of a growth-coupled lipid synthesis. An increase in specific growth rate (μ) from 0.03 to 0.05 h−1 resulted in a two-fold increase in the specific rate of lipid synthesis (milligram lipid (gram per lipid-free biomass) per hour). Under batch cultivation in glucose-containing media with urea or potassium nitrate as nitrogen sources, the ARA content was 46.0 and 60.4% of lipid; 16.4 and 18.8% of dry biomass; and 4.2 and 4.5 g l−1, respectively. Under continuous cultivation of the strain, the productivity of ARA synthesis was 16.2 and 19.2 mg l−1 h−1 at μ=0.05 and 0.03 h−1, respectively.

Biosynthesis of arachidonic acid by micromycetes (review)

Arachidonic acid (ARA, 5,8,1l,14-cis-eicosatetraenoic acid) is widely used in medicine, pharmaceutics, cosmetics, dietary nutrition, agriculture, and other fields. Microbiological production of ARA is of increased interest since the natural sources (pig liver, adrenal glands, and egg-yolk) cannot satisfy its growing requirements. Mechanisms for ARA biosynthesis as well as the regulation of enzymes involved in this process are considered. Review summarizes literature data concerning individual stages of microbiological ARA production, methods for screening of active strains-producers, physiological regulation of ARA synthesis in micromycetes (the effect of growth phase, medium composition, pH, temperature, and aeration), and effective technologies of fermentation and the product recovery. Information on the whole biotechnological process from strain selection to the ARA yield improvement and purification of the end product is presented.

[Growth-coupled lipid synthesis in Mortierella alpina LPM 301, a producer of arachidonic acid].

Mortierella alpina LPM 301, a producer of arachidonic acid (ARA), was found to possess a unique property of intense lipid synthesis in the period of active mycelium growth. Under batch cultivation of this strain in glucose-containing media with potassium nitrate or urea, the bulk of lipids (28–35% of dry biomass) was produced at the end of the exponential growth phase and remained almost unaltered in the stationary phase. The ARA content of lipids comprised 42–50% at the beginning of the stationary phase and increased continuously after glucose depletion in the medium due to the turnover of intracellular fatty acids; by the end of fermentation (189–210 h), the amount of ARA reached 46–60% of the total fatty acids (16–19% of dry mycelium). Plausible regulatory mechanisms of the growth-coupled lipid synthesis in microorganisms are discussed.

The effect of lipid content on the elemental composition and energy capacity of yeast biomass.

Oleaginous yeasts (18 strains) were grown in ethanol media under various cultivation conditions (33 biomass samples). It was found that lipid and lipid-free fractions of dry biomass have elemental composition and biomass reductivity very close to values which can be considered as biological constants. The energy content of dry biomass strongly depended on the total lipid content. When the lipid content was 64%, the energy value of dry biomass reached 73% of diesel oil; therefore, oleaginous microorganisms can be a promising source of biodiesel fuel. The approach used in this work makes it possible to determine the energy value of biomass by its elemental composition without application of laborious and expensive calorimetric measurements of combustion heats.

Kinetic characteristics of metal-EDTA degradation by immobilised cells of bacterial strain DSM 9103

Abstract A biosensor approach was applied to study the degradation of ethylenediaminetetraacetate (EDTA) and metal–EDTA complexes with Mg 2+ , Mn 2+ , Ba 2+ , and Ca 2+ by bacterial cells of strain DSM 9103 immobilised by sorption on Whatman GF/A chromatographic paper. Kinetic characteristics of EDTA degradation: maximum rate of oxygen consumption ( V max ), saturation constant ( K s ), and inhibitory constant ( K i ) were determined from the measured values of oxygen consumption rate at different substrate concentrations. Values of V max for all the substrates studied were similar whereas the values of K s and K i differed considerably. Uncomplexed EDTA and Ca–EDTA had the highest affinity to the EDTA-degrading system; Mn–EDTA showed the highest inhibitory effect. Results obtained offer considerable scope for the development of microbial biosensors sensitive to extremely low concentrations (5–100 μM) of EDTA.

Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg2+, Ca2+, Mn2+, Pb2+, Co2+, Cd2+, Zn2+, Cu2+, or Fe3+. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 1016 (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.

Arachidonic acid as an elicitor of the plant defense response to phytopathogens

Review summarizes both literature and own experimental data on the application of arachidonic acid (AA, C20:4 omega-6) as an elicitor of the plant defense reactions to phytopathogens. The elicitation activity of AA was shown to depend considerably on its concentration. High AA concentrations (over 10-5 M) induce necrosis of plant tissues and accumulation of antimicrobial compounds (phytoalexins), whereas low AA concentrations (10-7–10-7 M) elicit systemic and prolonged resistance to the phytopathogen infections that is similar to immunization process. Biochemical mechanisms responsible for the elicitor activity of AA involve the whole complex of reactions including reconstruction of the cell ultrastructure, an increase in the amounts of certain enzymes and protective substances, a decrease in sterol content, redirection of isoprenoid biosynthesis from sterol derivatives toward sesquiterpenoid phytoalexins, and appearance of signal molecules, which are spread all over the plant tissues making them immunized. Field experiments revealed that the treatment of potato, tomato, sugar beet, and vine plants with low concentrations of pure AA or AA-containing preparations isolated from Phytophtora and Mortierella fungi increased plant resistance to diseases (late blight, common scrab, rhizoctonoise, cercosporose, powdery mildew, etc.) and thus enhanced the harvest yield. The application of AA-containing preparations is an alternative to the use of environmentally dangerous chemical fungicides. The processes of microbiological AA production from renewable inexpensive raw substrates are considered.

Degradation of EDTA by a chemostat culture of bacterial strain DSM 9103

Abstract Characteristics of growth and EDTA degradation by chemostat culture of bacterial strain DSM 9103 were investigated. The effect of specific growth rate ( μ ) on mass cell yield ( Y X/S ), specific rate of EDTA uptake ( q S ) and the rate of Mg–EDTA degradation by a cell suspension ( q S rest ) was studied. Based on the experimental data, kinetic constants of dependencies q S ( S ) and μ ( S ) were determined. The rates of substrate and energy expenditures for cell maintenance, m S and m e , were 0.02 and 0.014 h −1 , respectively. High efficiency of DSM 9103 growth on EDTA in a chemostat was established; the maximum mass cell yield ( Y X/S m ) and the maximum energy cell yield ( η X/S m ) were as high as 35.3 and 50.5%, respectively. Results indicated that oxidation of side chains in the EDTA molecule by monooxygenase is coupled with energy utilization by the cells. Cells harvested from a chemostat (resting cells) retain the capability of EDTA degradation, although at a rate about four to five times lower than growing cells.

The effect of pH, aeration, and temperature on arachidonic acid synthesis by Mortierella alpina

The effects of pH, aeration, and temperature on the growth of fungal strain Mortierella alpina LPM-301 and the synthesis of lipids and arachidonic acid in glycerol-containing medium were studied. Arachidonic acid production in the stationary growth phase was found to depend considerably on the pH value; it reached the optimum at pH 6.0 and was irreversibly inhibited at a pH of 3.0. The pO2 values in a range from 10 to 50% showed no marked effect on mycelium growth or the synthesis of lipids and arachidonic acid. The temperature optimum for arachidonic acid production was 20–22°C. Under continuous cultivation, the amount of arachidonic acid reached 29.8% of lipids and 7.4% of biomass. The arachidonic acid yield from the glycerol consumed was 4.1% by mass and 8.8% by energy. It is suggested that arachidonic acid synthesis at an unfavorable pH and elevated temperatures was limited by the activity of Δ-12-desaturase and by the conversion of linoleic to arachidonic acid, respectively.

Bacterial strain characterizing by EDTA requirement

A novel strain of bacteria (LPM-4) characterized by a unique EDTA requirement for cell growth was isolated. Suspensions of washed cells of strain LPM-4 degraded EDTA complexes with Ba2+, Mg2+, Ca2+, and Mn2+ at constant rates ( 0.310 ± 0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 ± 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrading bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095− 1) and lower mass cell yield (0.219 g cells/g EDTA), which is promising for its practical applications for EDTA removal in wastewater treatment plants.