Milan Dostálek

Effect of culture conditions on mycelial growth and production of γ-linolenic acid by the fungus Mortierella ramanniana

SummaryEffect of culture conditions on cell growth, lipid accumulation and γ-linolenic acid production is reported for four Mortierella species. The highest concentration as well as the highest productivity of γ-linolenic acid in lipid was determined in strains of M. ramanniana. M. ramanniana CBS 112.08 was used in the studies of the influence of medium composition, concentration of carbon- and nitrogen sources and growth temperature. Several carbon sources provided good growth and a high lipid content in biomass. The highest dry weights (11–12g/l) and lipid contents (∼24%, w/w), were observed if glucose or fructose was used as carbon source, whereas the highest amount of γ-linolenic acid (∼26%) was determined in starch-grown cells. The fatty acid composition in the lipid was influenced by the cultivation time, growth temperature and, to a minor extent, by the carbon source used. In fermentor cultures, both strains of Mortierella ramanniana showed relatively poor growth and incomplete consumption of glucose. M. vinacea, on the other hand, grew well in tower reactors. M. vinacea, which has a different morphology than M. ramanniana strains, also showed higher yields of biomass and lipid and higher yield coefficients than the latter.

Biosurfactant production by Pseudomonas fluorescens 378: growth and product characteristics

SummaryAn isolate of Pseudomonas fluorescens, strain 378 was shown to produce a novel surface active compound (code name AP-6). The compound is unique in being a high molecular weight compound but has, in some aspects, properties of a low molecular weight surfactant. The product is extracellular and its formation appeared to be partly growth-associated. Using a semisynthetic medium, fermentor cultivations were performed in the pH range 6.8–8.4. The product yield was optimal at pH 8.0 and gave a final concentration of 210 times critical micelle dilution. At higher pH, specific growth rate, final biomass and product concentration decreased. It consists mainly of carbohydrates and protein, the molecular weight is 1×106 and the isoelectric point is pH 9.1.The surface tension of an aqueous solution reached 27 mN/m which is a very low value even compared to other surfactants of considerably lower size and the critical micelle concentration was less than 10 mg/l in 0.9% (w/v) NaCl. The kinetics of the adsorption process at the air-water interface was studied using the drop volume technique, and the reaction was found to be rapid, considering the size of the molecule. A concentration as low as 0.025 g/l reached a surface tension of 30 mN/m within 70 s.

Production of γ-linolenic acid by the fungus Mucor rouxii in fed-batch and continuous culture

SummaryThe production of γ-linolenic acid (GLA) and lipid was studied in Mucor rouxii CBS 416.77. In a fed-batch culture productivities of 39.4 mg/l per hour for GLA and 99 mg/l per hour for the total amount of lipid were determined at 18 h of cultivation. At this point the highest value of GLA in lipid (39.7%, w/w) was also reached. Production of GLA was also studied in a series of continuous cultures. It was observed that, in addition to growth rate, the nitrogen concentration of the input medium was of great importance for high productivities. The highest productivity values for GLA (37 mg/l per hour) and for lipid (95 mg/l per hour) were reached at a dilution rate of 0.10 h-1 with a concentration of 4.5g/l NH4Cl in the input medium.

Influence of cultivation conditions on lipid production by Cryptococcus albidus

SummaryCryptococcus albidus var. albidus CBS 4517 was able to accumulate lipid under nitrogen-limited as well as excess-nitrogen conditions. The highest lipid-producting capacity was, however, observed in nitrogen-limited cultivations. In nitrogen-limited batch cultures, a lipid content of 34% (w/w) in biomass and a maximum specific lipid productivity of 37 mg lipid/g lipid-free biomass·h, was determined. The yield of lipid from glucose was about 0.15 g/g in nitrogen-limited and 0.11 g/g in excess-nitrogen cultures.In a nitrogen-limited fed-batch culture, 12.4 g/l lipid was produced at 90 h of cultivation and the cells contained 46.3% (w/w) lipid.Higher lipid yield and cellular lipid content were observed when inorganic nitrogen sources were used compared with organic. The choice of carbon source was seen to influence growth as well as lipid production and the highest yields of lipid were obtained when glucose, maltose or mannitol was used.A cultivation temperature of 20°C provided the highest lipid productivity compared to 25°C and 30°C. Addition of citrate to the growth medium was seen to have a stimulating effect on the specific lipid productivity.

Mixed culture of Saccharomycopsis fibuliger and Zymomonas mobilis on starch-use of oxygen as a regulator

SummaryThe conversion of starch to ethanol in a mixed culture of an amylolytic yeast, Saccharomycopsis fibuliger and an anaerobic bacterium, Zymomonas mobilis, was studied. Interactions between the component cultures were commensalism and competition for glucose. Control of oxygen supply to the culture was used as an external regulator of growth and competition. No accumulation of reducing sugars was observed in the mixed culture when compared to a monoculture of Saccharomycopsis fibuliger grown on starch. The glucose formed was instantly used by Zymomonas mobilis for ethanol production and the glucose inhibition of hydrolysis of non-glucose reducting sugars was released. The final concentration of ethanol, 9.7 g·l−1, produced from 30 g·l−1 of starch, shows out that all the glucose available from starch hydrolysis was converted to ethanol. Glucose production from starch was the rate-limiting reaction in the system, causing a lower ethanol production rate in the mixed culture than in the monoculture of Zymomonas mobilis, grown on glucose.

Effect of culture conditions on fatty acid composition in lipids produced by the yeastcryptococcus albidus var. albidus

The influence of culture conditions on the fatty acid composition in lipids produced by Cryptococcus albidus var. albidus CBS 4517 was studied.The major fatty acids in C. albidus var. albidus were oleic (18:1), linoleic (18:2) and palmitic (16:0) acid. The relative amounts of fatty acids produced varied considerably during growth and lipid accumulation phases in nitrogen-limited as well as excess-nitrogen cultures. The degree of unsaturation correlated to the lipid content in the biomass and decreased with increasing amounts of cellular lipid. After glucose exhaustion, no further changes in the fatty acid composition nor in the lipid content of the cells were observed. A number of carbon and nitrogen sources could be utilized for lipid synthesis, but they influenced the fatty acid composition only to a minor extent.

Lipid formation by Cryptococcus albidus in nitrogen-limited and in carbon-limited chemostat cultures

SummaryCryptococcus albidus var. Albidus CBS 4517 was grown in nitrogen-limited and in carbon-limited chemostat cultures. The effect of growth rate and limiting nutrient on lipid accumulation and fatty acid composition was investigated.The maximum lipid content in the biomass was, in both cultivation systems, observed at the lowest dilution rate (growth rate) tested. At this dilution rate, D=0.31 h-1, cells from the nitrogen-limited culture contained 41% (w/w) lipid and cells from the carbon-limited culture 37%. These results indicate the ability of C. albidus, unlike other oleaginous yeasts, to accumulate lipid also in carbon-limited chemostats.The yield of lipid from carbon source was about the same at D=0.031 h-1 in nitrogen-limited (YL/S=0.16 g/g) as in carbon-limited (YL/S=0.17 g/g) cultures and decreased with increasing growth rates. In the nitrogen-limited culture, the lipid productivity was about constant at low growth rates (0.031–0.056 h-1) and a slight decrease was observed at D=0.08 h-1, while the specific lipid productivity, qL, increased to 27.5 mg/g per hour. In the carbon-limited culture, however, lipid productivity increased with increasing growth rates and reached its maximum value near μmax, whereas qL was about constant at 20 mg/g per hour.The fatty acid composition was influenced by the specific growth rate in nitrogen-limited as well as in carbon-limited cultures, although the changes were more pronounced during carbonlimitation. A decrease in the degree of unsaturation (Δ/mole) was also observed with increasing lipid content in the cells.

Production of lipid from starch by a nitrogen-controlled mixed culture of Saccharomycopsis fibuliger and Rhodosporidium toruloides

SummaryProduction of lipid from starch in a two-component mixed culture in a fed batch process was studied. Saccharomycopsis fibuliger was used as the amylolytic microorganism and Rhodosporidium toruloides, which does not grow on starch, as the lipid producer. The concentration of nitrogen can be used as an external regulator of growth and competition in this process. The total biomass concentration decreased and the relative amount of Rh. toruloides increased with decreasing initial concentration of nitrogen in the medium. The highest lipid concentration (9.7 g·l-1), highest overall lipid production rate (0.15 g·l-1·h-1) and highest concentration of lipid in biomass (36.5%) were obtained in cultures with an initial nitrogen concentration of 0.5 g·l-1. Compared to monocultures of Lipomyces starkeyi and Aspergillus oryzae on starch, the mixed culture showed slightly lower conversion of starch to lipid but was superior in the final lipid concentration and the overall lipid production rate.

Regulation of a mixed culture of Streptococcus lactis and Saccharomycopsis fibuliger

SummaryIn a mixed culture of Saccharomycopsis fibuliger Y76 and Streptococcus lactis 65.1 on starch the main interactions are commensalism and competition. Oxygen-limited batch and continuous cultures of S. fibuliger showed accumulation of sugars. When oxygen was used as an external regulatory parameter in the mixed culture lactic acid, acetic acid and formic acid were formed. Ethanol produced by S. lactis was most likely assimilated by S. fibuliger. Continuous mixed cultures were stable under conditions of oxygen limitation at the dilution rates tested (0.10 h−1 and 0.15 h−1). Conversion yields of 35 to 40% were obtained but may be improved.

Estimation of the biofilm formation ability of Pseudomonas putida in discrete samples from continuous culture

SummaryTest systems were set up in order to evaluate the ability of biomass from a continuous culture to form biofilms. A film-forming strain of Pseudomonas putida was used as the test organism. The adsorption of resting cells onto glass surfaces was measured in specially designed chambers containing 1 ml of cell suspension. Both the quantity and the physiological activity of the adsorbed cells, in terms of optical density after detachment and pH change of a substrate exposed to the adsorbed cells, were measured. The analysis of biomass from continuous cultures of Pseudomonas putida verified the suitability of the methods. Furthermore, other properties of importance to biofilm formation such as hydrophobicity and flocculation capacity of the cells were investigated.It was shown for samples deriving from different dilution rates that the cell adsorption rate drastically increased at dilution rates higher than the μmax of the culture. Simultaneously, higher values of hydrophobicity and flocculation capacity were observed.It was also shown that the age and thickness of the biofilm subsequently produced in the continuous culture influenced the metabolic activity per unit of biomass attached to the surface. The methods described in this investigation may facilitate the study of parameters important to biofilm formation as well as the metabolic activity of the attached biomass.