A Novel Olive Oil Degrading Thermoactinomyces Species with a High Extremely Thermostable Lipase Activity

A filamentous, Gram‐positive, spore forming aerobic bacterium was isolated from olive oil contaminated soil (Al Koura, Lebanon) on rhodamine agar plates at 60 °C. The isolate, HRK‐1 produced large quantities of an extracellular thermostable lipase which degrades olive oil. It was primarily classified as a Thermoactinomyces sp. due to the filamentous structure of its cells that bear one spore each on an un‐branched sporophore, the resistance of its spores to boiling, utilisation of sucrose as a carbon source and production of dark pigments. The isolate grew optimally at a temperature of 60 °C, a pH of 7.3 and an orbital shaking of 250 rpm. It showed an efficient olive oil degrading ability. No traces of triolein were detected after a 36‐h cultivation. A concentration of 10 % [v/v] olive oil did not inhibit its growth. Lipase production was constitutive, and did not depend on the presence of olive oil. The optimum concentration of olive oil for lipase activity was 1 % [v/v], and the activity was not enhanced at higher concentrations, but on the contrary, a decrease in enzyme activity was recorded. The lipase of HRK‐1 was preliminarily characterised in the crude cell‐free supernatant with a specific activity of 0.14 U/mg. It has an optimum activity at 60 °C and a pH of 8.0. This lipolytic enzyme showed resistance to boiling and to a wide range of metallic ions and inhibitors. The formation of this heat‐stable lipase started in the early exponential growth phase, while a maximum extracellular enzyme activity was detected at the end of the decline phase, when most of the cells appeared as spherical spores. The exceptionally high activity of lipase (2.37 U/ml) produced by HRK‐1 measured in the cell free supernatant clearly indicated the commercial importance of this isolate, especially after it showed great stability at elevated temperatures.     

Factorial designs: an efficient approach to choosing the main factors influencing growth and hydrolase production by <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

A fractional factorial design with eight trials was applied to select and model the effects of major factors, individually and in combination, on improving Tetrahymena thermophila growth and enzyme production. Regulated pH at 6.8 and olive oil at 0.5% (v/v) showed positive effects on fermentation, and increased cell growth parameters including generation time and maximal population formation. Lipase and protease production were also improved by these factors and were favoured by cultivation of Tetrahymena in darkness. This statistical experiment offers a beneficial and rapid screening procedure to select the most effective combination of factors influencing fermentation processes.     

Damaging agitation intensities increase DNA synthesis rate and alter cell-cycle phase distributions of CHO cells

The effects of fluid-mechanical force (agitation) on the cell cycle kinetics of Chinese hamster ovary (CHO) cells cultured in suspension in 2-L bioreactors has been examined. A two-color flow cytometry method was used to determine the fraction rate of DNA synthesis. With increased agitation intensity, cell viability decreased as a result of increased cell death. However, increased agitation induced the viable cells of the culture to a higher proliferative state relative to a control culture. The fraction of viable cells of the high-agitation culture (250 rpm) in S phase was higher (up to 45%) and in G1 phase was lower (up to 50%) compared with the viable cells of the control culture (80 rpm). The DNA synthesis rate per viable S-phase cell of the high-agitation culture was confirmed by recovery experiments, which were conducted to measure the apparent specific growth rate and the cell cycle kinetics of the high-agitation culture upon reduction in the agitation rate from 250 rpm back to 80 rpm. The apparent specific growth rate of the test culture, calculated for the first 12 h of the recovery period, was greater than the apparent specific growth rate of the control culture. Furthermore, the proliferative state of the viable cells of the test culture, which had become higher relative to the control culture during the high agitation period, gradually approached the level of the control culture during recovery. Results also show that the magnitude of the agitation intensity; the culture agitated at 250 rpm attained a greater proliferative state than a parallel culture agitated at 235 rpm. The 250-rpm culture had a higher fraction of S-phase and a lower fraction of G1-phase cells than the 235-rpm culture. The DNA sunthesis rate per viable S-phase cell of the 250-rpm culture was greater than of the 235-rpm culture. (c) 1992 John Wiley & Sons, Inc.     

Mass production of lipase by fed-batch culture of Pseudomonas fluorescens

Summary High concentration production of an extracellular enzyme, lipase, was achieved by a fed-batch culture of Pseudomonas fluorescens. During the cultivation, temperature, pH and dissolved oxygen concentration wwre maintained at 23°C, 6.5 and 2–5 ppm, respectively. Olive oil was used as a carbon source for microbial growth. To produce lipase effectively the specific feed rate of olive oil had to be maintained in a range of 0.04–0.06 (g oil) · (g dry cell)^-1 · h^-1. The CO₂ evolution rate was monitored to estimate the requirement of olive oil. The ratio of feed rate of olive oil to the CO₂ evolution rate was varied in the range of 20–60 g oil/mol CO₂. The higher value of the ratio accelerated microbial growth, but did not favour lipase production. Once the high cell concentration of 60 g/l had been achieved, the ratio was changed from 50 to 30 g oil/mol CO₂ to accelerate the lipase production. By this CO₂-dependent method a very high activity of lipase, 1980 units/ml, was obtained. Both the productivity and yield of lipase were prominently increased compared with a conventional batch culture.     

Effect of lard oil,olive oil and castor oil on oxygen transfer in an agitated fermentor

Adding lard oil, olive oil or castor oil into a baffled and agitated fermentor decreased the volumetric oxygen transfer coefficient (K_La) in the low concentration range. However, K_La was increased when concentration reached 0.25% (v/v) at 400 rpm. Experimental results indicated that 1% (v/v) olive oil was as good as 0.1% (v/v) polypropylene glycol when added to a yeast fermentation.     

Paraffin oil as a “methane vector” for rapid and high cell density cultivation of <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

Slow growth and relatively low cell densities of methanotrophs have limited their uses in industrial applications. In this study, a novel method for rapid cultivation of Methylosinus trichosporium OB3b was studied by adding a water-immiscible organic solvent in the medium. Paraffin oil was the most effective at enhancing cell growth and final cell density. This is at least partially due to the increase of methane gas transfer between gas and medium phases since methane solubility is higher in paraffin than in water/nitrate minimal salt medium. During cultivation with paraffin oil at 5% (v/v) in the medium, M. trichosporium OB3b cells also showed higher concentrations of the intermediary metabolites, such as formic acid and pyruvic acid, and consumed more methane compared with the control. Paraffin as methane vector to improve methanotroph growth was further studied in a 5-L fermentor at three concentrations (i.e., 2.5%, 5%, and 10%). Cell density reached about 14 g dry weight per liter with 5% paraffin, around seven times higher than that of the control (without paraffin). Cells cultivated with paraffin tended to accumulate around the interface between oil droplets and the water phase and could exist in oil phase in the case of 10% (v/v) paraffin. These results indicated that paraffin could enhance methanotroph growth, which is potentially useful in cultivation of methanotrophs in large scale in industry. Bing Han and Tao Su contributed equally to this work.     

Culture of microalgae Chlorella minutissima for biodiesel feedstock production

Microalgae are among the most promising of non‐food based biomass fuel feedstock alternatives. Algal biofuels production is challenged by limited oil content, growth rate, and economical cultivation. To develop the optimum cultivation conditions for increasing biofuels feedstock production, the effect of light source, light intensity, photoperiod, and nitrogen starvation on the growth rate, cell density, and lipid content of Chlorella minutissima were studied. The fatty acid content and composition of Chlorella minutissima were also investigated under the above conditions. Fluorescent lights were more effective than red or white light‐emitting diodes for algal growth. Increasing light intensity resulted in more rapid algal growth, while increasing the period of light also significantly increased biomass productivity. Our results showed that the lipid and triacylglycerol content were increased under N starvation conditions. Thus, a two‐phase strategy with an initial nutrient‐sufficient reactor followed by a nutrient deprivation strategy could likely balance the desire for rapid and high biomass generation (124 mg/L) with a high oil content (50%) of Chlorella minutissima to maximize the total amount of oil produced for biodiesel production. Moreover, methyl palmitate (C16:0), methyl oleate (C18:1), methyl linoleate (C18:2), and methyl linolenate (C18:3) are the major components of Chlorella minutissima derived FAME, and choice of light source, intensity, and N starvation impacted the FAME composition of Chlorella minutissima. The optimized cultivation conditions resulted in higher growth rate, cell density, and oil content, making Chlorella minutissima a potentially suitable organism for biodiesel feedstock production. Biotechnol. Bioeng. 2011;108: 2280–2287. © 2011 Wiley Periodicals, Inc.     

Lipase location in Yarrowia lipolytica cells

Lipase production by Yarrowia lipolytica was growth associated and, at the beginning of cultivation, it was mainly cell-bound. Lipase release into the culture medium started when about 50% of the carbon source (olive oil or glucose) was consumed reaching its maximum concentration in the late stationary phase.     

Optimization of protein-production by the baculovirus expression vector system in shake flasks

Summary Shake flasks were successfully employed for the cultivation of Spodoptera frugiperda (Sf-9) insect cells and for the production of \-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. The culture doubling time and maximal cell density were 20 h and 5 × 10⁶ cells/ml respectively. The optimal liquid volumes for flasks rotating at 100 rpm were 25–40% of the flask total volume. Enzyme production (about 600 mg/l) was best at a multiplicity of infection of between 1 and 20 and at a cell density at time of infection of 0.7 × 10⁶ cells/ml. At a rotation speed of 100 rpm, Pluronic F-68 had no effect on growth and enzyme production. Offprint requests to: Y. Shoham     

Use of moving aeration membrane bioreactor for the efficient production of tissue type plasminogen activator in serum free medium

A moving aeration-membrane (MAM) bioreactor was employed for the production of 2 μg/mL of tissue type Plasminogen Activator (tPA) in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating conditions, shear stress was as low as 0.65 dynes/cm² at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2 μg tPA/mL while maintaining a high cell density of 1.0×10⁷ viable cells/mL.     

Utilization of substrates during batch growth of Pseudomonas fluorescens on olive oil,lard, and mutton tallow

Summary Similar amounts of cell-associated and cell-free lipase activities were present in log phase cultures of Pseudomonas fluorescens growing on fatty substrates. The rates of triglyceride hydrolysis and fatty acid utilization were balanced as free fatty acids remained at low concentrations in the culture media at all times. The time required for growth initiation and optimal growth rates of P. fluorescens were similar on olive oil, lard and mutton tallow, provided the coalesced solid fats were mechanically dispersed during the initial stages of cultivation. Although olive oil was completely consumed at the end of cultivation, substantial amounts of tri- and diglycerides, presumably bearing saturated acyl residues, remained in lard culture and, to a lesser extent, in mutton tallow culture.     

Growth stimulation of a Shiro-like,mycorrhiza forming,mycelium of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> on solid substrates by non-ionic surfactants or vegetable oil

The incorporation of Tweens (1 %, 2 %, 5 %) or olive oil (1 %, 2 %) in soil or in soil-containing substrate strongly stimulated mycelial growth of the edible ectomycorrhizal mushroom Tricholoma matsutake (Matsutake) after 1 or 3 months, respectively. The growth responses to Tween 40 and Tween 80 were dose-dependent. Fungal biomass increased up to 15-fold as a result of olive oil incorporation. After 4 months of Matsutake/pine co-culture in the presence of olive oil (2 %), compact aggregates of substrate, hyphae, and surface-colonized roots were observed, recalling in some ways the mycelial mat structure of Matsutake in the field, i.e. Shiro. Olive oil did not prevent formation of well-developed Hartig net palmettis although those seemed rather less abundant than without oil addition. The incorporation of Tween 80 or olive oil (2 %) into nutrient agar induced the proliferation of peripheral hydrophilic-like hyphae penetrating the medium. Tricholoma matsutake growth stimulation, possibly related to the presence of fatty acids in surfactants and oil, could be a consequence of the higher hydrophilicity of treated hyphae, or of enhanced lytic enzyme excretion and activity. Parameters such as adjuvant type, concentration, and growth conditions will be further optimised to formulate culture substrates adapted to the co-culture of T. matsutake and its host plants.     

Isolation of a yeast growing on sardine oil and its characteristics

A yeast strain, FO-144Cl, was isolated from a soil sample, using crude sardine oil, which contains a large quantity of poly-unsaturated long-chain fatty acids, as a sole carbon source. This strain was identified as a species of Candida. A medium for its growth was optimized by statistical methods and optimal temperature for the growth was from 28 to 30°C. Among the natural oils and fats tested, the yeast grew best on olive oil and grew better on the crude sardine oil than on a refined one. The yield of dry cells was 17.6 mg/ml after 24 h, using 2% crude sardine oil. The maximum growth rate was 0.36, 0.25, and 0.21 h⁻¹ with crude sardine oil, soybean oil, and olive oil, respectively. The content of crude fat in the yeast cells was 15.1% and half of the total cell lipid was triglyceride. Fatty acid compositions of the lipid and oily fractions left in the medium after cultivation were analyzed. Little unsaturated long-chain fatty acids (>C₁₈) was observed in the cell lipids, but they were left concentrated in the medium.     

Production of lipase of Aspergillus foetidus in a batch stirred reactor

Summary Maximum lipase production byAspergillus foetidus was obtained from cultures grown in the medium of 2% olive oil and 0.5% sucrose. The optimal conditions for the production of lipases in the Multigen fermenters were found to be at 500rpm with an airflow of 1.5 liter per mimute. Immobilization of the fungal source was found to be infeasible in natural polymers.     

Lipid storage compounds in marine bacteria

Forty psychrophile or psychrotrophic crude-oil-utilizing marine bacteria were investigated for their ability to accumulate lipid storage compounds in the cytoplasm during cultivation under nitrogen-limiting conditions. Most of them (73%) were able to accumulate specialized lipids like polyhydroxyalkanoic acids (PHA) while other lipids such as wax esters occurred in two isolates. Accumulation of PHA occurred predominantly at low temperatures (4–20 °C) as demonstrated for three isolates. Electron microscopy revealed polyphosphate inclusions occurring in two isolates in addition to PHA. Cells of the isolate Acinetobacter sp. 211 were able to synthesize and accumulate lipid inclusions during growth on acetate, ethanol, olive oil, hexadecanol and heptadecane. The composition of the lipid inclusions depended on the compounds provided as carbon source. Wax esters and acylglycerols occurred mainly during the cultivation on olive oil; in contrast, wax esters and free alcohols occurred during cultivation on hexadecanol. Total fatty acids in cells of the Acinetobacter sp. 211 amounted to 25% of the cellular dry weight in olive-oil-grown cells. Palmitic acid was the main fatty acid in the lipids when the cells were cultivated on acetate or ethanol (44% and 32% of total fatty acids respectively). In contrast, fatty acids occurring in the lipids during cultivation on hexadecanol, heptadecane or olive oil were related to the carbon source. The fatty acids present in the accumulated lipids consisted predominantly of saturated and unsaturated straight-chain fatty acids with a chain length ranging from 12 to 18 carbon atoms. Analysis of the lipid-granule-associated proteins in cells of Acinetobacter sp. 211 revealed a protein of 39 kDa as the predominant protein species. Received: 2 July 1996 / Received revision: 3 September 1996 / Accepted: 28 September 1996     

A novel nicotinamide adenine dinucleotide control strategy for increasing the cell density of Haemophilus parasuis

Haemophilus parasuis is the causative agent of Glässer's disease and is a major source of economic losses in the swine industry each year. To enhance the production of an inactivated vaccine against H. parasuis, the availability of nicotinamide adenine dinucleotide (NAD) must be carefully controlled to ensure a sufficiently high cell density of H. parasuis. In the present study, the real-time viable cell density of H. parasuis was calculated based on the capacitance of the culture. By assessing the relationship between capacitance and viable cell density/NAD concentration, the NAD supply rate could be adjusted in real time to maintain the NAD concentration at a set value based on the linear relationship between capacitance and NAD consumption. The linear relationship between cell density and addition of NAD indicated that 7.138 × 10⁹ NAD molecules were required to satisfy per cell growth. Five types of NAD supply strategy were used to maintain different NAD concentration for H. parasuis cultivation, and the results revealed that the highest viable cell density (8.57, OD₆₀₀) and cell count (1.57 × 10¹⁰ CFU/mL) were obtained with strategy III (NAD concentration maintained at 30 mg/L), which were 1.46- and 1.45- times more, respectively, than cultures with using NAD supply strategy I (NAD concentration maintained at 10 mg/L). An extremely high cell density of H. parasuis was achieved using this NAD supply strategy, and the results demonstrated a convenient and reliable method for determining the real-time viable cell density relative to NAD concentration. Moreover, this method provides a theoretical foundation and an efficient approach for high cell density cultivation of other auxotroph bacteria.     

Enhancement of BDNF production by co-cultivation of human neuroblastoma and fibroblast cells

It has been proved that co-cultivation of human neuroblastoma cells and human fibrolast cells can enhance nerve cell growth and the production of BDNF in perfusion cultivation. In batch co-cultivation, maximum cell density was increased up to 1.76×10⁶ viable cells/mL from 9×10⁵ viable cells/mL of only neuroblastoma cell culture. The growth of neuroblastoma cells was greatly improved by culturing both nerve and fibroblast cells in a perfusion process, maintaining 1.5×10⁶ viable cells/mL, which was much higher than that from fed-batch cultivation. The nerve cell growth was greatly enhanced in both fed-batch and perfusion cultivations while the growth of fibroblast cells was not. It strongly implies that the factors secreted from, human fibroblast cells and/or the environments of co-culture system can enhance both cell growth and BDNF secretion. Specific BDNF production rate was not enhanced in co-cultures; however, the production period was increased as the cell growth was lengthened in the co-culture case. Competitive growth between nerve cells and fibroblast cells was not observed in all cases, showing no changes of fibroblast cell growth and only enhancement of the neuroblastoma cell growth and overall BDNF production. It was also found that the perfusion cultivation was the most appropriate process for cultivating two cell lines simultaneously in a bioreactor.     

Production of rosmarinic acid by<Emphasis Type="Italic">Lavandula vera</Emphasis> MM cell suspension in bioreactor: effect of dissolved oxygen concentration and agitation

The relationship between dissolved oxygen (DO) concentration, agitation rate and growth of Lavandula vera MM and rosmarinic acid biosynthesis was investigated in 3 l laboratory bioreactor. Lavandula vera MM cell suspension accumulated the highest amounts of biomass (34.8 g/l) and rosmarinic acid (1870.6 mg/l) on day 12 of cultivation at 50% dissolved oxygen and agitation speed 100 rpm and at 30% dissolved oxygen and agitation speed 300 rpm, respectively.     

Edible mushrooms from olive oil mill wastes

The biological remediation of olive oil mill wastes has been attempted several times in the past through the use of different types of microbes. Among them, a relatively large array of fungi were studied for neutralizing the heavy pollutant effects and/or for converting these wastes into new value-added products. The present investigation was aiming at examining whether olive oil mill wastes could be exploited for the cultivation of mushrooms of the genus Pleurotus. At a preliminary stage, two Pleurotus species, i.e. P. eryngii and P. pulmonarius, were tested for their ability to colonize an olive press-cake (OPC) substrate supplemented with various dilutions of raw olive mill wastewater (OWW). Some important cultural characters related to mushroom production (earliness, yield, biological efficiencies and quality of basidiomata) were estimated. The outcome revealed different cultural responses for each Pleurotus species examined; the P. pulmonarius strain showed better earliness values and P. eryngii, although it was a slow growing fungus, produced basidiomata in high yields and of a very good quality. On the other hand, the OPC substrate supplemented with low concentrations of OWW (12.5% v/w) behaved satisfactorily as regards the fungal colonization rates and mushroom yield, but when the addition of higher rates of raw, untreated OWW (75–100% v/w) was attempted then the Pleurotus strains were completely unable to grow. The optimal concentration of OWW for Pleurotus mycelial growth was assessed through measurements of the biomass produced in liquid nutrient media and was found to lie within the 25–50% range, depending on the Pleurotus species and on the properties of the substrates examined. Furthermore, the phytotoxic effects that the spent liquid medium possessed were examined in comparison with the phytotoxicity of the raw liquid waste. The prospects of exploiting olive oil mills wastes for mushroom cultivation is discussed.     

Influence of carbon and nitrogen sources on lipase production by a newly isolated Candida viswanathii strain

Microorganisms can produce lipases with different biochemical characteristics making necessary the screening of new lipase-producing strains for different industrial applications. In this study, 90 microbial strains were screened as potential lipase producers using a sensitive agar plate method with a suitable medium supplemented with Tween 20 and also a liquid culture supplemented with olive oil. The highest cell growth and lipase production for Candida viswanathii were observed in triolein and oleic acid when used as the only pure carbon source. Renewable low-cost triacylglycerols supported the best cell growth, and olive oil was found to be the best inducer for lipase production (19.50 g/L and 58.50 U). The selected conditions for enzyme production were found with yeast extract as nitrogen source and 1.5 % (w/v) olive oil (85.70 U) that resulted in a good cell growth yield (Y_X/S?=?1.234 g/g) and lipase productivity (1.204 U/h) after 72 h of shake-flask cultivation. C. viswanathii lipase presented high hydrolytic activity on esters bonds of triacylglycerols of long-chain, and this strain can be considered an important candidate for future applications in chemical industries.