Utilization of gas oil by a yeast culture

A mixed yeast culture (Culture 4) was grown on representative gas oil samples as well as paraffin wax. Culture 4 was found to utilize n-paraffinic hydrocarbons almost quantitatively from most gas oil fractions; significant alteration of other hydrocarbon components was not detected. Generation times of 4.0–9.0hr. were typical during the exponential growth phase in fermentations with various gas oil fractions. Cell yields were 70–90% based on n-paraffin utilization. The culture appeared to exhibit maximum efficiency of n-alkane removal in the C₁₉ to C₂₄ range. The cells recovered from the fermentations contained 8.8–9.3% nitrogen. Paraffin wax also served as a suitable carbon source when dissolved in 2,6,10,14-tertramethylpentadecane (pristane). However, substrate utilization appeared to be incomplete.     

Hydrocarbon fermentations using Candida lipolytica. I. Basic growth parameters for batch and continuous culture conditions

Candida lipolytica (strain ATCC 8661) was grown on a simple defined medium with n-dodecane as sole carbon source under batch and continuous fermentation conditions. The composition of cellular material recovered from the fermentations, the oxygen demand of the cells, and the effect of operating conditions on cell growth were evaluated experimentally. These basic data are presented and discussed.     

Bioremediation of Hydrocarbons in Contaminated Wood: A Proof‐of‐Concept Study

A proof‐of‐concept study to evaluate the biological removal of hydrocarbons (naphthalene, n‐hexadecane, and fuel oil #2) from contaminated wood (Southern yellow pine) was conducted using ¹⁴C‐labeled tracers and gas chromatography. Contaminated wood was brought in contact with n‐hexadecane‐degrading Pseudomonas aeruginosa PG201 or naphthalene degrading environmental isolates by the application either on mineral medium agar or filter paper containing a previously grown biomass (“overlay” technique). The experiments showed a significant acceleration of naphthalene removal by biomass. Due to biodegradation combined with evaporation, naphthalene was nearly completely removed (up to 90–98 %) in 4–8 days from freshly contaminated 6 mm‐ and 17 mm‐thick wood samples. The removal of a less volatile hydrocarbon, n‐hexadecane, was less efficient, at 40–60% in 20–40 days, with the only variable significantly affecting this pollutant's removal rate being the moisture content of the medium. Biodegradation experiments with standard heating fuel oil #2 (a representative real‐world contaminant) resulted in significant removal of light hydrocarbons (C₁₀–C₁₆), i.e., more mobile/volatile substrates, in 3 weeks (up to 70 %) whereas heavier hydrocarbons (C₁₇–C₁₉) were less affected. Pollutant mobility in both wood and aqueous media was shown to be the crucial factor affecting the removal efficiency. These results point toward a promising technique to reclaim wooden structures contaminated with volatile and semi‐volatile chemicals.     

Characterization of selected actinomycetes degrading polyaromatic hydrocarbons in liquid culture and spiked soil

Summary Five strains of the Rhodococcus and Gordonia genera were evaluated for their potential use in bioremediation of polycyclic aromatic hydrocarbons (PAH) with or without another substrate (co-substrate). Their ability to produce biosurfactants or to degrade phenanthrene when growing on glucose, hexadecane and rapeseed oil was tested in liquid medium at 30 °C. All strains showed biosurfactant activity. The highest reduction in surface tension was recorded in whole cultures of Rhodococcus sp. DSM 44126 (23.1%) and R. erythropolis DSM 1069 (21.1%) grown on hexadecane and Gordonia sp. APB (20.4%) and R. erythropolis TA57 (18.2%) grown on rapeseed oil. Cultures of Gordonia sp. APB and G. rubripertincta formed emulsions when grown on rapeseed oil. After 14 days of incubation, Rhodococcus sp. DSM 44126 degraded phenanthrene (initial concentration 100 μg ml⁻¹) as sole carbon source (79.4%) and in the presence of hexadecane (80.6%), rapeseed oil (96.8%) and glucose (below the limit of detection). The other strains degraded less than 20%, and then with a co-substrate only. Rhodococcus sp. DSM 44126 was selected and its performance evaluated in soil spiked with a mixture of PAH (200 mg kg⁻¹). The effect of the addition of 0, 0.1 and 1% rapeseed oil as co-substrate was also tested. Inoculation enhanced the degradation of phenanthrene (55.7% and 95.2% with 0.1% oil and without oil respectively) and of anthracene (29.2% with 0.1% oil). Approximately 96% of anthracene and 62% of benzo(a)pyrene disappeared from the soil (inoculated and control) after 14 days and anthraquinone was detected as a metabolite. Rhodococcus sp. DSM 44126 was identified as Rhodococcus wratislaviensis by 16S rRNA sequencing and was able to degrade anthracene as sole carbon source in liquid culture.     

Cellulase and protein production from mixed cultures of Trichoderma viride and a yeast

Fermentations with mixed cultures of the cellulolytic fungus Trichoderma viride and the yeast Saccharomyces cerevisiae or Candida utiliswere examined. The fermentations were carried out in an aerated 5 liter fermentor with NaOH treated barley straw as the cellulose source (2–4%). Yeast was inoculated 24–32 hr after the fungus and the growth of the two organisms was followed through the production of CO₂ and cell protein. In comparison with fermentations with T. viridealone, the production time for maximum yields of cellulases and cell protein was reduced by several days, depending on the straw concentrations. The protein content of the growth product was 21–22% and the amino acid composition of the product resembled that of T. viride alone.     

Occurrence of fatty alcohol oxidase in alkane-and fatty-acid-utilising yeasts and moulds

Preparations of membrane fractions from 16 yeasts and three moulds were assayed for long-chain fatty alcohol oxidase (FAOD) activities after being grown on hexadecane or glucose and, in nine cases, on oleic acid. The enzyme was usually repressed in glucose-grown cells but in Candida bombicola ATCC 22 214 and Debaryomyces hansenii NCYC 33 appeared to be constitutive. Highest activities occurred in C. tropicalis and D. polymorphus (about 0.8 unit/mg protein) grown on hexadecane. Growth of yeasts on oleic acid partially induced FAOD activity but not with the moulds. In two strains of Yarrowia lipolytica (DSM 3286 and CBS 2076) no activity of FAOD was found but this could have been due to the known photo-lability of the enzyme. FAOD from different species shared similar characteristics with respect to substrate specificity and pH optimum. Correspondence to: C. Ratledge     

Anaerobic oxidation of saturated hydrocarbons to CO2 by a new type of sulfate-reducing bacterium

n-Hexadecane added as electron donor and carbon source to an anaerobic enrichment culture from an oil production plant or to anoxic marine sediment samples allowed dissimilatory sulfate reduction to sulfide. The enrichment from the oil field was purified via serial dilutions in liquid medium under a hexadecane phase and in agar medium with caprylate. A pure culture of a sulfate-reducing bacterium, strain Hxd3, with relatively tiny cells (0.4–0.5 by 0.8–2 m) was isolated that grew anaerobically on hexadecane without addition of further organic substrates. Most of the cells were found to adhere to the hydrocarbon phase. It was verified that neither organic impurities in hexadecane nor residual oxygen were responsible for growth. Strain Hxd3 was grown with n-hexadecane of high purity (99.5%) in anoxic glass ampoules sealed by fusion. Of 0.4 ml hexadecane added per l (1.4 mmol per l), 90% was degraded with concomitant reduction of sulfate. Controls with pasteurized cells or a common Desulfovibrio species neither consumed hexadecane nor reduced sulfate. Incubation of cell-free medium with low reducing capacity and a redox indicator showed that the ampoules were completely oxygen-tight. Measured degradation balances and enzyme activities suggested a complete oxidation of the alkane to CO₂ via the carbon monoxide dehydrogenase pathway. However, the first step in anaerobic alkane oxidation is unknown. On hexadecane, strain Hxd3 produced as much as 15 to 20 mM H₂S, but growth was rather slow; with 5% inoculum, cultures were fully grown after 5 to 7 weeks. The new sulfate reducer grew on alkanes from C₁₂ to C₂₀, 1-hexadecene, 1-hexadecanol, 2-hexadecanol, palmitate and stearate. Best growth occurred on stearate (doubling time around 26 h). Growth on soluble fatty acids such as caprylate was very poor. Alkanes with chains shorter than C₁₂, lactate, ethanol or H₂ were not used. Strain Hxd3 is the first anaerobe shown to grow definitely on saturated hydrocarbons.Abbreviations CO dehydrogenase carbon monoxide dehydrogenase - DTE 1,4-dithioerythritol - Tris tris(hydroxymethyl)-aminomethane Dedicated to Dr. Ralph S. Wolfe on occasion of his 70th birthday     

Effect of culture conditions on batch growth of Saccharomycopsis lipolytica on olive oil

Summary Batch cultures of Saccharomycopsis lipolytica were grown in minimal medium with olive oil as carbon source. Inocula of glucose-grown cells commenced growth with little lag at rates largely unaffected by variations in the stirring rate or oil concentration. However, growth rates declined when the medium pH was below 7.0. In all cultures, media pH declined with increasing cell concentration. Cell composition during exponential growth was 42% protein and 2% fat. Carbon-limited cells maintained this composition after oil exhaustion but during nitrogen- and oxygen-limited growth, protein content decreased and fat content increased although the protein decrease was only transient with oxygen limitation. Yield coefficients for triglyceride were near unity for all cultures. Free acid concentrations rose rapidly after inoculation. As fermentations progressed, free glycerol appeared and concentrations of di- and monoglycerides passed through maximal values although peak concentrations of di- and monoglycerides persisted for extended times in oxygen- and nitrogen-limited cultures respectively. The fraction of free glycerol consumed was greater in oxygen-limited than in carbon- or nitrogen-limited culture. The basic requirements for growth of yeasts on fatty wastes are discussed with reference to these observations.     

Characteristics of hexadecane partition by the growth medium of Acinetobacter sp.

The partition of n-hexadecane in the spent growth medium of Acinetobacter sp. HOI-N was determined by measuring the increase in the relative aqueous solubility of ³H-hexadecane as compared to controls. The amount of hexadecane partitioned was proportional to the protein concentration. The specific solubility of hexadecane (nmol/mg protein) was analyzed by least-squares fitting yielding an average slope of 0.6 with a standard deviation of 0.3, indicating either nonequilibrium of hexadecane or physical aggregation of protein. The amount of hexadecane partitioned was concentration dependent yielding optically clear microemulsions at hexadecane concentrations of less than 1.4mM and macroemulsions at hexadecane concentrations of 1.4mM or greater. Preliminary results indicated that hexadecane and partitioned by a lipoprotein complex.     

Microbial response to crude oil and Corexit 9527: SEAFLUXES enclosure study

The response of marine bacteria to Corexit 9527, with and without Prudhoe Bay crude oil labeled withn–(1–¹⁴C)hexadecane, in a temperate pelagic environment was monitored over 22 days using controlled ecosystem enclosures. The results indicated that Corexit and Corexit-dispersed crude oil stimulated bacterial production by serving as substrates and/or by inducing the release of organic compounds from the indigenous phytoplankton population. Highest bacterial standing stock was observed in the enclosure treated with a mixture of Corexit and crude oil, in which a large fraction of the predominant bacterivores were eliminated. Biodegradation appeared to be more significant than abiotic processes in contributing to the loss of low volatility n-alkanes in Corexit-dispersed oil. Twenty-two days following its addition, 50% of the radiotracer was recovered: 3% in the suspended particulate fraction, 10% in sedimentary material, 36% as CO₂, and less than 1% in the dissolved organic pool.     

Development of an E. coli strain for cell-free ADC manufacturing

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody–drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a 5-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.     

Clutivation of yeast on gas oil

The results achieved by the cultivation of the yeast. Candida lipolytica on gas oil are referred. By using a distillation fraction of gas oil distilling between 180–400°C, containing 10–20% of n-alkanes, the optimal condition for biomass production and deparaffination were estimated for various dilution rates and various amounts of gas oil in the medium. The main factor, which influences the yield coefficient by hydrocarbon fermentation is the polyauxie of the hydrocarbon substrate. The penetration of dispersed hydrocarbons into the yeast cell is demonstrated on electron micrographs and the velocity and reversibility of this process is estimated by using tritium-traced hexadecane.     

Partition of hexadecane to the cell surface of Candida tropicalis: Mechanism for the transport of water-insoluble substrates

The partition of hexadecane to the cell surface of Candida tropicalis was measured by incubating heat-inactivated cells with hexadecane-1-¹⁴C on a gyratory shaker. The free hexadecane was separated by centrifuging the cells through a 15% sucrose solution, and the partitioned hexadecane was quantified by scintillation spectrometry of the samples from the resulting cell sediment. Heat-inactivated cells did not take up hexadecane as determined by a membrane filtration technique involving organic solvent washing. The partitioning was a time-dependent process. The velocity increased by increasing the shake rate of te shaker. At 360 rpm and with baffled flasks, saturation of the cell surface with hexadecane was obtained after a 20 min incubation period. The amount of hexadecane partitioned depended on the initial hexadecane-to-cell concentration ratio. At a ratio of 5 μmol/mg cell protein the highest amount of hexadecane partitioned was measured at 2100 μmol/mg cell protein. At ratios higher than 6 μmol/mg cell protein the cells were no longer sedimentable by centrifugation. The partition of hexadecane to the cell surface was affected by removing the surface layer of the cell wall by Pronase treatment and by using detergents in the partition assay. Pronase treatment lowered the amount of hexadecane partitioned as a consequence of the removal of the lipophilic layer of the cell surface. Detergents influence the partition coefficient and also lowered the amount of hexadecane partitioning to the cell surface. At a low shaking intensity (280 rpm, unbaffled flasks), after Pronase treatment, and in the presence of detergents he uptake of hexadecane by the cells was limited by the partitioning.     

Growth models of cultures with two liquid phases. 8. Experimental observations on droplet size and interfacial area

Because of the importance of the drop she distribution and interfacial area of the dispersed liquid phase in hydrocarbon fermentations, experiments were carried out to determine the drop size distribution and the interfacial area during batch fermentations of Candida lipolytica on gas oil and on n-hexadecane dissolved in dewaxed gas oil. The effects of cell concentration and dispersed phase volume fraction on size distribution and interfacial area were investigated. Measurements of interfacial tensions, densities, viscosities, and fatty acid concentrations were also made. The results show that the size distribution is skewed and that the Sauter mean diameter is in the range of 10 to 30 μ. Both the Sauter mean diameter and the interfacial area increased during the course of a batch fermentation; however, they decreased at the end of the fermentation. The interfacial area also increased with inoculum size.     

Biodegradation of plasticizers by Rhodococcus rhodochrous

Rhodococcus rhodochrous was grown in the presence of oneof three plasticizers: bis 2-ethylhexyl adipate (BEHA), dioctyl phthalate (DOP) ordioctyl terephthalate (DOTP). None of the plasticizers were degraded unless anothercarbon source, such as hexadecane, was also present. When R. rhodochrous was grownwith hexadecane as a co-substrate, BEHA was completely degraded and the DOP was degraded slightly. About half of the DOTP was degraded, if hexadecane were present.In all of these growth studies, the toxicity of the media, which was assessed usingthe Microtox assay, increased as the organism degraded the plasticizer. In each case, therewas an accumulation of one or two intermediates in the growth medium as the toxicityincreased. One of these was identified as 2-ethylhexanoic acid and it was observed forall three plasticizers. Its concentration increased until degradation of the plasticizershad stopped and it was always present at the end of the fermentation. The other intermediatewas identified as 2-ethylhexanol and this was only observed forgrowth in the presence of BEHA. The alcohol was observed early in the growth studies with BEHA and haddisappeared by the end of the experiment. Both the 2-ethylhexanol and 2-ethylhexanoicacid were shown to be toxic and their presence explained the increase of toxicity asthe fermentations proceeded. The appearance of these intermediates was consistent with similar degradation mechanisms for all three plasticizers involving hydrolysisof the ester bonds followed by oxidation of the released alcohol.     

Production and release of surfactant byCorynebacterium lepus in hydrocarbon and glucose media

Summary Corynebacterium lepus produced a considerable amount of extracellular surfactant during growth in a mineral salts medium containing hexadecane as the sole carbon source. The study revealed that the bacterium also produced a large amount of surfactant when grown on glucose, but in this case the surface active agent was cell bound. The surfactant was released from the cells when they were treated with hexadecane after growth. Tetradecane also showed a good capability for release of the surfactant. Decane and octane were less effective than hexadecane and tetradecane.     

Microbial transformation of hydrocarbons. II. Growth constants and cell composition of microbial cells derived from n-alkanes

Cultivation of Norcardia sp., Mycobacterium phlei, and Candida lipolytica in inorganic salt solution containing n-alkanes C₁₀–C₂₀ as solo carbon and energy source was investigated. Generation times of 0.5–7.0 hr were typical during the exponential growth phase. The final cell concentrations (dry weight) were usually 9–26 g/l with n-alkane mixtures ranging from n-decane through n-eicosane. A linear dependence was found between the production of cell mass and the consumption of n-alkanes. The rest concentration of n-alkanes in the cell mass is in all experiments smaller than 0.5% (w/w). Cell yields were Y_sub 60–142% and for Y_e 50–97% based on n-alkane utilization. In one case, with the Nocardia NBZ 23, the substrate specifity on hydrocarbons and on a n-alkane mixture C₁₀-C₂₀ was studied. The cell mass recovered from the fermentations contained 47.8–57.7% carbon, 5.6–9.95% nitrogen, 7.2–9.4% hydrogen, 35–62% crude protein, and 6–36% lipid. Cellular protein and lipid synthesized by an organism is influenced by the type of nitrogen source. The amino acid, glucosamine, muramic acid, 2,6-diaminopimelinic acid, and fatty acid distribution in organisms grown on n-alkanes compared with a corresponding fermentation on glucose as sole carbon source were also estimated.     

Cell surface properties of five polycyclic aromatic compound-degrading yeast strains

To investigate the effects of physiological properties on polycyclic aromatic compound (PAH) degradation, the surface tension and emulsification activities, and cell surface hydrophobicity of five PAH-degrading yeast isolates were compared to Saccharomyces cerevisiae from cultures grown with glucose, hexadecane, or naphthalene as carbon sources. The cell surface hydrophobicity values for the five yeast strains were significantly higher than for S. cerevisiae for all culture conditions, although these were highest with hexadecane and naphthalene. Strains with higher hydrophobicity showed higher rates of naphthalene and phenanthrene degradation, indicating that increased cell hydrophobicity might be an important strategy in PAH degradation for the five strains. Emulsification activities increased for all five yeast strains with naphthalene culturing, although no relationship existed between emulsification activity and PAH degradation rate. Surface tensions were not markedly reduced with naphthalene culturing.     

Lytic Enzymes for Gram-negative Bacteria Produced by Bacillus subtilis YT-25

The available energy, gross protein value, phosphorus availability and palatability of 16 samples of single cell protein were evaluated in 20 bioassays using total 2,136 depleted chicks.

Four protein samples were products from Aspergillus tamarii grown on waste water of a fish processing factory, three were from Aspergillus oryzae grown on either acetic acid medium or cooked soybean waste, three were from Candida sp. grown on citrus molasses extracted from peel wastes of citrus processing plants, four were from Candida utitis grown on wood molasses produced from various wood wastes, and two were from Pseudomonas sp. and Alteromonas thlasomethanolica grown on methanol.

Five of 16 samples had excellent nutritive value, comparable to single cell proteins available commercially in Europe. All samples were palatable to the chicks, and no sign of acute toxicity was observed.     

Cell Surface Measurements in Hydrocarbon and Carbohydrate Fermentations

Acinetobacter calcoaceticus was grown in 11-liter batch fermentations with hexadecane or sodium citrate as the sole source of carbon. Surface and interfacial tension measurements of the microbial broth indicated that surface-active compounds were being produced only during growth on the hydrocarbon substrate. Contact angle measurements of an aqueous drop on a smooth lawn of cells in a hexadecane bath indicated a highly hydrophobic surface of the cells in the initial stages of the hydrocarbon fermentation (120° contact angle). At this stage, the entire cell population was bound to the hydrocarbon-aqueous interface. The contact angle dropped rapidly to approximately 45° after 14 h into the fermentation. This coincided with a shift of the cell population to the aqueous phase. Thus, the cells demonstrated more hydrophilic characteristics in the later stages of the fermentation. Contact angles on cells grown on sodium citrate ranged from 18 to 24° throughout the fermentation. The cells appear to be highly hydrophilic during growth on a soluble substrate. From the contact angle and aqueous-hydrocarbon interfacial tension, the surface free energy of the cells was calculated along with the cell-aqueous and cell-hydrocarbon interfacial tension. The results of these measurements were useful in quantitatively evaluating the hydrophobic nature of the cell surface during growth on hydrocarbons and comparing it with the hydrophilic nature of the cell surface during growth on a soluble substrate.