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.     

A Hypothetical Cyclic Pathway for the Metabolism of Odd-carbon n-Alkanes or Propionyl-CoA via Seven-carbon Tricarboxylic Acids in Yeasts

An extensive study has been undertaken to elucidate the physiological significance of threo-D_s-2-methylisocitric acid produced mainly from odd-carbon n-alkanes by a mutant strain of Candida lipolytica. The mutant strain showed slower growth responses to odd-carbon n-alkanes, especially of shorter chain-length, and failed to utilize this acid as sole carbon source, whereas the parent strain and many other yeasts tested were able to utilize this acid. About one half of yeasts tested accumulated this acid extracellularly. Under a thiamine-deficient condition, amounts of pyruvate produced by the parent strain from odd-carbon n-alkanes were ten times as large as those from even-carbon n-alkanes. A scheme for the partial oxidation of propionyl-CoA to pyruvate via C₇-tricarboxylic acid by yeasts was supposed. This scheme may offer suggestion on the metabolism of propionyl-CoA by other living organisms. A hypothetical pathway of citrate accumulation from odd-carbon n-alkane was also presented.     

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.     

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.     

Effects of Chain-length of Alkane Substrate on Fatty Acid Composition and Biosynthetic Pathway in Some Candida Yeasts

Cellular fatty acid compositions of Candida tropicalis pK 233 and Candida lipolytica NRRL Y -6795 and the time-course changes during yeast growth were studied using individual n-alkanes of various chain lengths (from C₁₁ to C₁₈) and a mixture of n-alkanes (C₁₁ to C₁₈) as a sole carbon source. Observed relationships of the chain-length of n-alkane substrate to time-course changes and final patterns of the fatty acid compositions of these yeasts, especially those of the cells grown on odd-carbon alkanes, indicated that “intact incorporation mechanism,” that is, accumulation of the fatty acid having the same chain-length as that of the alkane substrate used was predominant in the yeasts cultivated on a longer alkane such as n-heptadecane and n-octadecane. On the other hand, “chain elongation pathway” and “de novo synthesis pathway” following β-oxidation of substrate were simultaneously operative in the cells growing on a relatively shorter alkane such as undecane and dodecane.     

Fermentation of n-Paraffins by Yeast

Nutritional requirement of Candida lipolytica AJ 5004 and its productivity of α-ketoglutarate were further studied.

It became clear that this yeast required only thiamine as grown factor, and even if the yeast was cultured in chemically defined medium containing adequate amount of thiamine, it was able to produce as high yield of α-ketoglutarate as in the medium containing 0.02% of corn steep liquor.

It was also shown that the rate of convertion of n-paraffin to α-ketoglutarate gradually increased as the concentration of n-paraffins was decreased or as the incubation time was prolonged. A very high rate of conversion, 71%, was obtained after prolonged culture, for 5 days, with a culture medium containing 8% of n-paraffins.

The productivity of α-ketoglutarate from C₉- to C₂₀-alkanes by the yeast was maximum in the range from C₁₅ to C₁₉, especially from C₁₇ to C₁₉.     

Candida lipolytica mutants defective in an acyl-CoA synthetase

Summary Mutant strains of Candida lipolytica NRRL Y-6795, which are defective in fatty acyl-CoA synthetase I linking to the system incorporating the fatty acyl moiety into cellular lipids (Kamiryo, et al., 1977), were cultivated on various carbon sources including odd-chain n-alkanes (C₁₁ to C₁₇) and their fatty acid compositions were examined.In the case of the wild-type strain grown on odd-chain n-alkanes (from C₁₃ to C₁₇), the proportions of odd-chain cellular fatty acids to total cellular fatty acids were markedly high, reaching 98–99% in the n-pentadecane- and n-heptadecane-grown cells. Those of the mutant strains, however, were drastically low, being at most 12–13% even in the n-heptadecane-grown cells. The total fatty acid contents in the mutant cells were 4–5% in dry weight, being slightly lower than those of the wild strain (4–7% in dry weight).The growth rates of the mutants on glucose, n-undecane and n-tridecane were comparable to those of the wild strain. When n-pentadecane, n-heptadecane, or oleic acid was used as carbon source, the mutants had lower, but still practicable, growth rates.The results obtained indicate that these mutant strains of Candida lipolytica will be useful as sources of biomass with low content of nonnatural odd-chain fatty acids.     

Isolation and Identification of n-Butane-assimilating Bacterium

A bacterial strain capable of assimilating gaseous n-alkanes was newly isolated from activated sludge by enrichment culture technique using n-butane as the sole carbon source. The strain was identified as Pseudomonas butanovora sp. nov. It utilised n-alkanes of C₂~C₉, primary alcohols and carboxylic acids for growth, but did not utilize sugars and C₁ compounds. The cell yields on gaseous n-alkanes, such as ethane, propane and n-butane, were 80% or more. The maximum specific growth rate on n-butane was 0.22 hr^?1 at 30°C, pH 7.0. Dried cells of this new isolate grown on n-butane contained 73% pure protein.     

The substrate specificity of two yeast strains utilizing hydrocarbons

StrainsCandida lipolytica 4-1 andCandida lipolytica K were compared in their growth and dawaxing capacities during batch growth on model gas oil. The model gas oil was composed of a mixture of even-numbered puren-alkanes (n-decane ton-dotriacontane) dissolved in dewaxed gas oil. The results show that both strains differ in their substrate specificity and in the sequence of utilization of individualn-alkanes. Strain K, previously used for dewaxing of mineral oil, has its substrate specificity shifted toward the highern-alkanes.     

Degradation of long-chain n-alkanes by the yeast Candida maltosa

Summary The yeast Candida maltosa precultivated on liquid n-alkanes utilized different solid n-alkanes (especially C₂₀–C₂₅) in the presence of pristane as an organic phase with rates comparable to, or somewhat larger than, those of liquid n-alkanes. Analysis of cellular fatty acids indicated an assimilation of solid n-alkanes via monoterminal oxidation. The resulting fatty acids with substrate chain length were chain-shortened by C₂ units down to an optimal range of chain length from C₁₆ to C₁₈ and incorporated into cellular, lipids directly or after desaturation. The intermediates of chain-shortening with numbers of carbon atoms higher than C₁₈, as well as the unusually long-chain fatty acids of substrate chain length, were detected in trace amounts only. Even-carbon-numbered and odd-numbered fatty acids predominated in experiments with evenchain and odd-chain n-alkanes, respectively. Studies with cerulenin indicated that de novo synthesis of fatty acids was negligible. Oxidation of solid n-alkanes by the yeast C. maltosa yielded fatty acid patterns similar to those of cells grown on liquid n-alkanes.     

Some Specific Features of the Respiration of Hydrocarbon-utilizable Candida Yeasts

The respiration of both glucose-grown and hydrocarbon-grown cells of Candida tropicalis pK 233 harvested in the stationary phases was not inhibited by cyanide when glucose was used as oxidation substrate, but the former was rather stimulated in the presence of cyanide. When n-alkanes were used as oxidation substrate, cyanide lowered the respiratory activities of both cells to about 50%. With respect to the susceptibility to cyanide, the younger cells growing on n-alkanes were less sensitive in hydrocarbon oxidizing ability than the older cells, whereas the older cells growing on glucose or n-alkanes were more resistant in glucose oxidizing ability than the younger cells. Acetate was oxidized by both glucose-grown and hydrocarbon-grown cells of the yeast. Laurate was oxidized by hydrocarbon-grown cells, but not by glucose-grown cells. The respiration on laurate was inhibited completely by 3.3 mM of cyanide. In general, hydrocarbon-grown cells of Candida tropicalis pK 233 were more sensitive to various respiratory inhibitors than glucose-grown cells, although the oxidation substrates had a significant effect.

The respiration of both glucose-grown and hydrocarbon-grown cells of C. albicans, C. guilliermondii and C. lipolytica harvested in the stationary phases was also resistant to cyanide when glucose was used as oxidation substrate. But the respiration on n-alkanes of these cells was inhibited significantly by 3.3 mM of cyanide except for C. albicans.     

Growth of Candida albicans in the presence of hydrocarbons: a correlation between sterol concentration and hydrocarbon uptake

Summary Candida albicans KTCC 89062 grown on n-alkanes showed higher levels of sterol content as compared to glucose-grown cells. Certain sterols, such as lanosterol, were significantly reduced in cells grown on n-alkanes, while others, such as ergosterol, increased in these cells. Sterol fractions declined as the chain length of the n-alkanes increased. Ergosterol supplementation of the chemically defined medium showed an increase in the uptake of dodecane (C₁₂) by cells grown on such medium. Increase in the concentration of ergosterol supplementation resulted in an increase in C₁₂ uptake. The uptake of C₁₂ was not stimulated by ergosterol supplementation in the case of non-viable yeast cells.     

<Emphasis Type="Italic">n</Emphasis>-Alkanes variability in the diazotrophic cyanobacterium <Emphasis Type="Italic">Anabaena cylindrica</Emphasis> in response to NaCl stress

n-Alkanes pattern in response to NaCl stress has been studied in the cyanobacterium Anabaena cylindrica. Saturated hydrocarbons were separated and identified by gas chromatography-mass spectrometry (GC-MS) using serially coupled capillary column. Light chain n-alkanes in the range of C₉–C₁₇ (43%) and heavy chain n-alkanes in range of C₁₇–C₂₃ (34%) and C₂₃–C₃₁ (23%) were identified as the major components of total hydrocarbons in the NaCl adapted cells of A. cylindrica. In contrast, NaCl-untreated cells of A. cylindrica had dominance of only long chain n-alkanes in the range of C₂₃–C₃₁ comprising about 94% of its total n-alkanes. The persistence of high level (43%) of short chain n-alkanes (C₉–C₁₇) in NaCl adapted cells of A. cylindrica as compared to its negligible level (0.2%) in NaCl untreated counterpart clearly indicates that NaCl stress causes the A. cylindrica to shift towards the synthesis of short chain n-alkanes.     

Effects of biosurfactants produced by Candida antarctica on the biodegradation of petroleum compounds

The effects of biosurfactants on the biodegradation of petroleum compounds were investigated. Candida antarctica T-34 could produce extracellular biosurfactant mannosylerythritol lipids (MELs) when it was cultured in vegetable oil. In addition, in our previous study, it was found that this strain could also produce a new type of biosurfactant while it grew on n-undecane (C₁₁H₂₄), and the biosurfactant was named as BS-UC. In flask culture of Candida antarctica, the addition of BS-UC could improve the biodegradation rate of some n-alkanes (e.g. 90.2% for n-decane, 90.2% for n-undecane, 89.0% for dodecane), a mixture of n-alkanes (82.3%) and kerosene (72.5%). By comparing the effects of the biosurfactants BS-UC and MEL and chemical surfactants on the biodegradation of crude oil, it was found that biosurfactants could be used to enhance the degradation of petroleum compounds instead of chemical surfactants. In a laboratory scale immobilized bioreactor, the addition of biosurfactant improved not only the emulsification of kerosene in simulated wastewater but also its biodegradation rate. The highest degradation rate of kerosene by addition of MEL and BS-UC reached 87 and 90% at 15 h, respectively. The results showed that the biosurfactant BS-UC was highly promising for work on biodegradation of hydrophobic contaminants.     

Cultivation of the yeast candida lipolytica on hydrocarbons. I. Degradation of n-alkanes in batch fermentation of gas oil

The growth of batch-cultivated yeast Candida lipolytica on three kinds of gas oil using mineral medium was studied. A linear dependence was found between the production of yeast biomass and the consumption of n-alkanes, while the decrease of freezing point of gas oil during cultivation had a distinct course. This disproportion was explained by different degradation of individual n-alkanes contained in gas oil. The rate of degradation of pentadecane, hexadecane, and heptadecane was the same during the entire cultivation. On the contrary, in the first phase the utilization of shorter chain n-alkanes, nonane to tetradecane, was more rapid while that of longer chain homologs, octadecane to pentacosane, lagged. Rapid utilization of longer chain n-alkanes did not occur before the concentration of the other n-alkanes decreased. Only then the rapid decrease of freezing point appeared.     

Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases

A bacterial strain capable of utilizing n-alkanes with chain lengths ranging from decane (C₁₀H₂₂) to tetracontane (C₄₀H₈₂) as a sole carbon source was isolated using a system for screening microorganisms able to grow on paraffin (mixed long-chain n-alkanes). The isolate, identified according to its 16S rRNA sequence as Acinetobacter venetianus, was designated A. venetianus 6A2. Two DNA fragments encoding parts of AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, were polymerase chain reaction-amplified from the genome of A. venetianus 6A2. To study the roles of these two alkM paralogues in n-alkane utilization in A. venetianus 6A2, we constructed alkMa, alkMb, and alkMa/alkMb disruption mutants. Studies on the growth patterns of the disruption mutants using n-alkanes with different chain lengths as sole carbon source demonstrated central roles for the alkMa and alkMb genes in utilization of C10 to C18 n-alkanes. Comparative analysis of these patterns also suggested different substrate preferences for AlkMa and AlkMb in n-alkane utilization. Because both single and double mutants were able to grow on n-alkanes with chain lengths of C20 and longer, we concluded that yet another enzyme(s) for the utilization of these n-alkanes must exist in A. venetianus 6A2.     

Fermentation of n-Paraffins by Yeast

α-Ketoglutarate productivity from n-paraffins of 141 strains of identified yeasts was studied. Among the strains tested, only strains of Candida lipolytica exclusively showed a high ability to produce α-ketoglutarale.

It was also observed that these strains of Candida lipolytica required thiamine for their growth and that exegenous thiamine stimulated the activity of α-ketoglutarate dehydrogenase of Candida lipolytica AJ 5004.

From these results, relationship between thiamine requirement and α-ketoglutarate productivity of Candida lipolytica was discussed.

α-Ketoglutarate fermentation by representative strains of Candida lipolytica was also carried out.     

Biomarker indications for microbial contribution to Recent and Late Jurassic carbonate deposits

Summary Biomarker investigations were applied to the hydrocarbon fractions of three Recent (cyanobacterial mat, Lake Van microbialite and Lake Satonda microbialite) and two Late Jurassic carbonate samples obtained from sponge bioherms. The relative concentrations ofn-alkanes, monomethyl alkanes, acyclic isoprenoids, steroids and hopanoids in these samples are studied and their probable biological precursors are discussed. Normal alkanes with carbon chain lengths ranging from C₁₅ to C₃₄ and monomethyl alkanes ranging from C₁₇ to C₂₁ with a varying methyl branching pattern are found. The major hydrocarbons are low molecular weight (LMW)n-alkanes (C₁₅–C₂₁) with a slight to strong predominance ofn-heptadecane (C₁₇). High molecular weight (HMW)n-alkanes occur in low to moderate relative concentrations showing a preference of odd-carbon numbered compounds with a maximum at C₂₉. Within the acyclic isoprenoids, pristane, phytane/phytene, pentamethyl-eicosane, squalane and lycopane could be identified. Polycyclic terpenoids of the sterane and/or hopane type are present in all carbonate samples. The carbon numbers of these components range from 27 to 29 and 27 to 32, respectively. These organic compounds identified can be attributed to various source organisms such as cyanobacteria, archaebacteria, algae and vascular plants. All hydrocarbon fractions of the samples are characterized by moderate to high relative concentrations of compounds derived from cyanobacteria, signifying the role of these organisms as contributors to the Recent as well as to the Late Jurassic carbonate deposits.     

Cultivation of the yeast Candida lipolytica on hydrocarbons. II. One-stage continuous cultivation on gas oil

Continuous cultivation of the yeast Candida lipolytica on gas oil was studied from the viewpoint of biomass production and oil deparaffination. Optimum conditions wore found at the dilution rate D = 0.16–0.19 when biomass productivity 1.7 g/l/hr and yield coefficient. y = 0.92 were achieved. At deparaffination to the same freezing point, more than double the production of biomass and deparaffined oil during a given time unit was achieved in a continuous process than in batch cultivation. Consumption of substrate was followed in both cultivation processes and it was confirmed that individual n-alkanes of gas oil were degraded at various rates and yields. Results proved optimum cultivation conditions to depend on concentration and composition of the paraffinic fraction of gas oil used. To achieve these conditions the continuous process may be controlled by choice; of suitable dilution rate and concentration of gas oil.     

Fermentation of n-Paraffins by Yeast

Screening test for obtaining microorganisms which produce l-amino acids or organic acids from n-paraffins were carried out. Fourteen strains of microorganisms which seemed to belong to the yeast showed strong ability to produce α-ketoglutaric acid. A representative strain of these microorganisms was identified as Candida lipolytica AJ 5004.

Optimal conditions for production of α-ketoglutarate using Candida lipolytica AJ 5004 were also studied. Under the condition thus obtained using a culture medium of 8 weight % of n-paraffins, the yeast accumulated 59% of α-ketoglutarate to the substrate added after three days culture.