Biodegradation of C5-C8 fatty acids and production of aroma volatiles by Myroides sp. ZB35 isolated from activated sludge

In the effluents of a biologically treated wastewater from a heavy oil-refining plant, C₅-C₈ fatty acids including pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, and 2-methylbutanoic acid are often detected. As these residual fatty acids can cause further air and water pollution, a new Myroides isolate ZB35 from activated sludge was explored to degrade these C₅-C₈ fatty acids in this study. It was found that the biodegradation process involved a lag phase that became prolonged with increasing acyl chain length when the fatty acids were individually fed to this strain. However, when fed as a mixture, the ones with longer acyl chains were found to become more quickly assimilated. The branched 2-methylbutanoic acid was always the last one to be depleted among the five fatty acids under both conditions. Metabolite analysis revealed one possible origin of short chain fatty acids in the biologically treated wastewater. Aroma volatiles including 2-methylbutyl isovalerate, isoamyl 2-methylbutanoate, isoamyl isovalerate, and 2-methylbutyl 2-methylbutanoate were subsequently identified from ZB35 extracts, linking the source of the fruity odor to these esters excreted by Myroides species. To our best knowledge, this is the first finding of these aroma esters in bacteria. From a biotechnological viewpoint, this study has revealed the potential of Myroides species as a promising source of aroma esters attractive for food and fragrance industries.     

Lipids of the Green Alga BotryococcusCultured in a Batch Mode

The lipid fraction of the green alga Botryococcuscultured in a batch mode was found to contain polar lipids (more than 50% of the total lipids), di- and triacylglycerols, sterols and their esters, free fatty acids, and hydrocarbons. In aging culture, the content of polar lipids somewhat decreased and that of triacylglycerols increased by more than four times. The content of hydrocarbons in the algal biomass did not exceed 0.9% and depended little on the culture age. Intracellular lipids contained saturated and unsaturated (mono-, di-, and trienoic) fatty acids. The maximum content of C_{16 : 3}and -C_{18 : 3}fatty acids (up to 35% of the total fatty acids) was detected in the phase of active growth. The extracellular and intracellular lipids of the alga differed in the proportion of particular lipids and in the fatty acid pattern.     

Preparation of extracts of culture liquids for gas-chromatographic determination of acidic fermentation products

An extraction method coupled with gas-chromatographic analysis has been devised for the determination of small amounts of C₁-C₇ branched and straight-chain saturated fatty acids as well as 18 mono-, di- and tricarboxylic acids and related compounds, the latter being analyzed either as their methylesters or as their trimethylsilyl-derivatives. Methods for extracting non-volatile acids or both volatile and non-volatile acids are described for large and small volumes of culture media. The proposed methods have been successfully applied to the determination of non-acidic fermentation products, known amounts of alcohols, and to volatile and non-volatile acids in bacterial fermentation solutions, namely a batch culture ofLactobacillus casei subsp.rhamnosus ATCC 7469. A carbon balance sheet of this organism's metabolic behaviour throughout growth is proposed.     

Distribution of C22-, C24- and C26-α-Unit-Containing Mycolic Acid Homologues in Mycobacteria

There are three mycolic acid homologues with C₂₂-, C₂₄- and C₂₆-α-units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non-polar fatty acids (free and bound forms), we have separated non-polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin-layer chromatography, and analyzed non-polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B-29]⁺ ions (loss of CHO from the α-branched-chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C₂₂-, C₂₄- and C₂₆-α-units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060-1070, 1986). By pyrolysis GC, C_22:0, C_24:0 and C_26:0 fatty acid methyl esters generated by the C₂-C₃ cleavage of C₂₂-, C₂₄- and C₂₆-α-unit-containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non-polar C_22:0, C_24:0 and C_26:0 fatty acids. By mass chromatography, the composition and distribution of C₂₂- and C₂₄-α-unit-containing homologues were revealed to be similar between α- and α'-mycolic acids in every Mycobacterium. We further analyzed in detail M. vaccae and demonstrated that the mass chromatogram of C₂₂-α-unit-containing homologue was analogous in shape to that of the C₂₄-α-unit-containing one, with the latter mass chromatogram being up-shifted from the former by two carbon numbers, in every subclass of α-, α'-, keto and dicarboxy mycolic acids. The present study suggests that the compositions of three homologues of both mycolic acids and constitutive non-polar fatty acids, which are characteristic to each mycobacterial species, may reflect the proportion of the amount of free C_22:0, C_24:0 and C_26:0 fatty acids synthesized in the cell. It is further demonstrated that intermolecular condensation of two fatty acids which become α- and β-units of mycolic acids will occur independently of the carbon chain length or kinds of polar moieties of fatty acids.     

Ester Production by Pseudomonas fragi. II. Factors Influencing Ester Levels in Milk Cultures

Cultures of Pseudomonas fragi were grown at 21 C in sterile homogenized milk and reconstituted skim milk media supplemented with ethyl alcohol. Quantitative determinations of ethyl butyrate and ethyl hexanoate by gas-liquid chromatography showed definite increases in the concentrations of the two esters produced in these media in comparison to media not supplemented with ethyl alcohol. Supplementation with butyric acid in addition to ethyl alcohol generally elevated the ethyl butyrate concentration and usually depressed the cell count slightly. Aeration of any of the media during growth tended to reduce the cell population slightly. A relationship between increase in cell number and increase in concentration of esters during the growth of the culture was observed. Media containing high concentrations of ethyl alcohol plus milk fat or low-molecular-weight fatty acids were conducive to the production of a fruity aroma by P. fragi.     

Formation of polysaccharide depolymerase and glycoside hydrolase enzymes byBacteroides ruminicola subsp.ruminicola grown in batch and continuous culture

The activity of ten polysaccharide depolymerase and glycoside hydrolase enzymes was monitored inBacteroides ruminicola subsp.ruminicola throughout the growth cycle and over a range of dilution rates in carbon-limited continuous (chemostat) culture. The enzymes were principally cell associated, and the specific activities increased throughout the growth cycle to reach maximum levels in the late exponential and stationary growth phases. In chemostat-grown cells the activities were growth rate dependent and were highest at the lowest dilution rates examined (0.025/h), but remained constant over a wide range of growth rates (D=0.05–0.15/h). The specific activities were lower in cells with a generation time of 3 h (D=0.225/h). The major metabolites formed from xylose, in batch and continuous culture, were lactic, acetic, and succinic acids, with traces (1%–2% of total acid production) of branched and straight-chain C₃–C₅ volatile fatty acids. The proportions of the metabolites produced varied with the stage and rate of growth.     

Fatty acids in green algae cultivated on a pilot-plant scale

Fatty acids fromChlorella vulgaris, Scenedesmus obliquus var.acutus and from a mixed culture of the two strains, Melnik, were converted to methyl esters, separated by gas chromatography, and identified by means of standards. The spectrum of fatty acids included both saturated and unsaturated acids (with odd and even numbers of carbon atoms) from C₁₂ to C₂₂. Fatty acids C_16:0, C_18:0 and C_20:3 were the major components in all cultures. Pure strains differed from the mixed culture in the production of C_18:1, C_12:0 and C_19:2 acids; the first of these was present in higher amounts in pure cultures only, the latter two being found in the mixed culture. The level of lipids was lower as compared to the literature data and their extractability was affected by the manner of preparation of algae and extraction conditions.     

Composition of the epicuticular and intracuticular wax layers on Kalanchoe daigremontiana (Hamet et Perr. de la Bathie) leaves

Epicuticular and intracuticular waxes from both adaxial and abaxial surfaces of the leaves of Kalanchoe daigremontiana were analyzed. All wax mixtures were found to contain approximately equal amounts of triterpenoids and very long chain fatty acid (VLCFA) derivatives. The triterpenoid fraction consisted of glutinol (8-19% of the total wax) and friedelin (4-9%), together with smaller amounts of glutanol, glutinol acetate, epifriedelanol, germanicol and β-amyrin. The VLCFA derivatives comprised C₂₇-C₃₅ alkanes (19-37% of the total wax), C₃₂-C₃₄ aldehydes (3-7%), C₃₂ and C₃₄ fatty acids (0.2-3%), C₂₆-C₃₆ primary alcohols (4-8%), and C₄₂-C₅₂ alkyl esters (2-9%). The wax layers were found to differ in triterpenoid amounts, with the intracuticular wax containing higher percentages of most triterpenoids than the epicuticular wax. Friedelin, the only triterpenoid ketone present, showed the opposite distribution with higher proportions in the epicuticular wax. VLCFA derivatives also accumulated to higher percentages in the epicuticular than in the intracuticular wax layer. Epicuticular wax crystals were observed on both the adaxial and abaxial leaf surfaces.     

Isolation and characterization of fatty acid methyl ester (FAME)‐producing Streptomyces sp. S161 from sheep (Ovis aries) faeces

An actinomycete producing oil‐like mixtures was isolated and characterized. The strain was isolated from sheep faeces and identified as Streptomyces sp. S161 based on 16S rRNA gene sequence analysis. The strain showed cellulase and xylanase activities. The ¹H nuclear magnetic resonance (NMR) spectra of the mixtures showed that the mixtures were composed of fatty acid methyl esters (52·5), triglycerides (13·7) and monoglycerides (9·1) (mol.%). Based on the gas chromatography–mass spectrometry (GC‐MS) analysis, the fatty acid methyl esters were mainly composed of C14‐C16 long‐chain fatty acids. The results indicated that Streptomyces sp. S161 could produce fatty acid methyl esters (FAME) directly from starch. To our knowledge, this is the first isolated strain that can produce biodiesel (FAME) directly from starch.

Significance and Impact of the Study

Nowadays, production of biodiesel is based on plant oils, animal fats, algal oils and microbial oils. Lipid mostly consists of triacylglycerols (TAG), and conversion of these lipids into fatty acid short‐chain alcohol esters (methanol or ethanol) is the final step in biodiesel production. In this study, an oil‐producing Streptomyces strain was isolated from sheep faeces. The oil was composed of C₁₄‐C₁₆ long‐chain fatty acid methyl esters, triglycerides and monoglycerides. This is the first isolated strain‐producing biodiesel (FAME) directly from starch. Due to showing cellulase and xylanase activities, the strain would be helpful for converting renewable lignocellulose into biodiesel directly.     

Surface wax of Andreaea and Pogonatum species

The mosses Andreaea rupestris, Pogonatum aloides and P. urnigerum contain surface waxes in amounts of 0.05–0.12% dry wt. The waxes consisted of esters (C₃₈-C₅₄), primary alcohols (C₂₀-C₃₂), free fatty acids (C₁₆-C₃₀), and alkanes (C₂₁-C₃₁). Additionally, aldehydes (C₂₂-C₃₀) were major constituents in the wax of P. urnigerum. The classes and their chain length distributions in the surface waxes of these mosses are comparable to those of epicuticular waxes of higher plants.     

Influence of fatty acids on the growth of wine microorganisms Saccharomyces cerevisiae and Oenococcus oeni

The effects of fatty acids, extracted during prefermentation grape skin-contact on Saccharomyces cerevisiae and Oenococcus oeni, were studied. The influence of skin-contact on total fatty acid content was evaluated both in Chardonnay must and in synthetic medium. Prior to alcoholic fermentation, the skin-contact contributes to a large enrichment of long-chain fatty acids (C₁₆ to C_18:3). These results induced a positive effect on yeast growth and particularly on cell viability. In the skin-contact fermented media, levels of C₁₂ and especially C₁₀ are lower and macromolecules content higher than in controls. This production of extracellular mannoproteins and the reduction of medium-chain fatty acids in media by S. cerevisiae increased growth of O. oeni. The influence of fatty acids (C₁₀ to C_18:3), in their free and esterified forms, on bacterial growth and on malolactic activity was also examined. Only C₁₀ and C₁₂, especially in their esterified forms, always appeared to be toxic to O. oeni. Received 15 May 1997/ Accepted in revised form 02 December 1997     

Stearic acid methyl ester: A new extracellular metabolite of the obligate methylotrophic bacterium Methylophilus quaylei

Methyl esters of fatty acids, free fatty acids, and hydrocarbons were found in the culture liquid and in the cellular lipids of the obligate methylotrophic bacterium Methylophilus quaylei under optimal growth conditions and osmotic stress. The main extracellular hydrophobic metabolite was methyl stearate. Exogenous free fatty acids C₁₆–C₁₈ and their methyl esters stimulated the M. quaylei growth and survivability, as well as production of exopolysaccharide under osmotic and oxidative stress, playing the role of growth factors and adaptogens. The order of hydrophobic supplements according to the ability to stimulate bacterial growth is C_{18: 1} > C_{18: 0} > C_{16: 0} > methyl oleate > methyl stearate > no supplements > C_{14: 0} > C_{12: 0}. The mechanism underlying the protective action of fatty acids and their methyl esters is discussed.     

Oleyl Oleate and Homologous Wax Esters Synthesized Coordinately from Oleic Acid by Acinetobacter and Coryneform Strains

Newly isolated Acinetobacter (NRRL B-14920, B-14921, B-14923) and coryneform (NRRL B-14922) strains accumulated oleyl oleate and homologous liquid wax esters (C_30:2–C_36:2) in culture broths. Diunsaturated oleyl oleate preponderated in 75 mg liquid wax esters (280 mg lipid extract) recovered from 100-ml cultures of Acinetobacter B-14920 supplemented with 810 mg oleic acid–oleyl alcohol. With soybean oil instead of oleic acid, wax esters (260 mg) were increased to approximately 50% of the lipid extract. Production of wax esters by cultures supplemented with combined fatty (C₈–C₁₈) alcohols and acids suggests a coordinated synthesis whereby the exogenous alcohol remains unaltered, and the fatty acid is partially oxidized with removal of C₂ units before esterification. Consequently, C₈–C₁₈ primary alcohols control chain lengths of the wax esters. Exogenous fatty acids are presumed to enter an intracellular oxidation pool from which is produced a homologous series of liquid wax esters.     

Occurrence and Carbon Metabolism of Green Nonsulfur-like Bacteria in Californian and Nevada Hot Spring Microbial mats as Revealed by Wax Ester Lipid Analysis

Green nonsulfur-like bacteria (GNSLB) in Yellowstone hot spring microbial mats have been extensively studied and are thought to operate both as photoheterotrophs and photoautotrophs. Here we studied the occurrence and carbon metabolisms of GNSLB by analyzing the distribution and isotopic composition of their characteristic wax ester lipids in four Californian and Nevada hot spring microbial mats at a range of temperatures (37–96°C). The distribution of wax esters varied strongly with temperature. At temperatures between 50–60°C the wax ester composition in each of the four hot spring microbial mats was dominated by C₃₀ to C₃₆ wax esters, consisting of mixtures of C₁₅-C₁₈ n-alkyl and branched fatty acids and alcohols, typical for GNSLB. Stable carbon isotopic analysis showed that these wax esters were only depleted by 5 to 10‰ compared to dissolved inorganic carbon in the overlying water, suggesting that these GNSLB were mainly autotrophic. However, analysis of different depth layers of one microbial mat showed that these GNSLB wax esters were increasingly depleted in ¹³C with depth, suggesting that photoautotrophy mainly occurred in the top layer of the mat. ¹³C-depleted C₃₆-C₄₄ wax esters were found in one hot spring at high temperatures (77–96°C) and are likely derived from allochtonous plant waxes. At several lower temperature sites (35–40°C) the wax esters were predominantly composed of C₂₈, C₃₀ and C₃₂ wax esters consisting of mixtures of C₁₄-C₁₆ fatty acids and n-alkanols and were depleted in ¹³C by 15–20‰ relative to dissolved inorganic carbon, suggesting they may be derived from heterotrophic organisms. Our results indicate that autotrophic GNSLB occur widely in hot springs and that diverse groups of organisms contribute to the pool of wax ester lipids in hot spring environments.     

Degradation of long chain n-alkanes by Chlorococcales

Summary Four out of thirty-one algae strains belonging to the order Chlorococcales exhibited good growth on solid media containing n-alkanes. Chlorella vulgaris (397) was able to degrade n-tridecane in cooxidation. The corresponding secondary alcohols and ketones in C₂-to C₇-position could be identified in the culture broth. The same oxidation products could be found in the media of cultures grown in darkness with the addition of glucose. This demonstrates a subterminal degradation pathway of C. vulgaris.There was no indication for a mono-or diterminal oxidation of alkanes by algae.The fatty acid pattern of lipids exhibited an incease in long chain acids and a decrease in shorter chain acids. The growth rate of cells grown on alkanes increased after 72 h, but the release of autospores was retarded.     

EFFECTS OF LOWERING TEMPERATURE DURING CULTURE ON THE PRODUCTION OF POLYUNSATURATED FATTY ACIDS IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The composition of fatty acids and contents of eicosapentaenoic acid (EPA) and polyunsaturated fatty acids (PUFAs) of the economically important marine diatom, Phaeodactylum tricornutum (Bohlin), were investigated to see whether reducing the culture temperature enhances the production of EPA and PUFAs. The contents of EPA and PUFAs of P. tricornutum were found to be higher at lower temperature when cultured at 10, 15, 20, or 25°C. When the cells grown at 25°C were shifted to 20, 15, or 10°C, the contents per dry mass of PUFAs and EPA increased to the maximal values in 48, 24, and 12 h, respectively. The highest yields of PUFAs and EPA per unit dry mass (per unit volume of culture) were 4.9% and 2.6% (12.4 and 6.6 mg·L^?1), respectively, when temperature was shifted from 25 to 10°C for 12 h, both being raised by 120% compared with the control. The representative fatty acids in the total fatty acids, when temperature was lowered from 25 to 10°C, decreased proportionally by about 30% in C_16:0 and 20% in C_16:1(n?7) but increased about 85% in EPA. It was concluded that lowering culture temperature of P. tricornutum could significantly raise the yields of EPA and PUFAs.     

Microbial fatty acid specificity

Strains ofRhodotorula sp.,Candida spp. andLangermania sp. cultivated on polyunsaturated oil preferentially incorporated more unsaturated fatty acids. These fatty acids were used mainly for growth needs whereas the saturated ones accumulated in the microbial cell. The cellular oil and the remaining oil in the culture had a lower degree of unsaturation as compared to the initial oil, and a modified fatty acid composition.Candida lipolytica, in a chemostat continuous culture, incorporated C₁₈ fatty acids in the order of C_18:3>C_18:2>C_18:1>C_18:0, and accumulated mostly the saturated ones. The specific productivity of the cellular oil and of the oil remaining in the culture medium was 0.036 and 0.487 gg⁻¹ h⁻¹, respectively, at dilution rateD=0.2/h.     

New chemicals from the dufour gland of the formicine ant Lasius niger (Hymenoptera:Formicidae)

A series of esters of C₈-C₁₃ fatty acids formed with C₁₀-C₁₂ straight chain alcohols, representing 3.7% of the glandular contents, was found in the Dufour gland of workers of the formicine ant Lasius niger. Altogether, nearly 50 components, among them hydrocarbons, alcohols, acetates and propionates were identified in the glandular liquid by means of gas chromatography (GC) and mass spectrometry (MS), using a solid injection technique. The wax esters and the propionates—decyl, dodecyl, octadecyl and farnesyl—have not been previously reported from ant secretions.     

Further evidence for an elongation-decarboxylation mechanism in the biosynthesis of paraffins in leaves

In isolated tobacco leaves l-valine-U-¹⁴C gave rise to labeled even-numbered isobranched fatty acids containing 16 to 26 carbon atoms and iso C₂₉, iso C₃₁, and iso C₃₃ paraffins. l-Isoleucine-U-¹⁴C on the other hand produced labeled odd-numbered anteiso C₁₇ to C₂₇ fatty acids and anteiso C₃₀ and C₃₂ paraffins. Trichloroacetic acid inhibited the incorporation of isobutyrate into C₂₀ and higher fatty acids and paraffins without affecting the synthesis of the C₁₆ and C₁₈ fatty acids. Thus the very long branched fatty acids are biosynthetically related to the paraffins. In Senecio odoris leaves acetate-1-¹⁴C was incorporated into the paraffins (mainly n-C₃₁) only in the epidermis although acetate was readily incorporated into fatty acids in the mesophyll tissue. Similarly only the epidermal tissue incorporated acetate into fatty acids longer than C₁₈ suggesting that the epidermis is the site of synthesis of both paraffins and the very long fatty acids. In broccoli leaves n-C₁₂ acid labeled with ¹⁴C in the carboxyl carbon and ³H in the methylene carbons was incorporated into C₂₉ paraffin without the loss of ¹⁴C relative to ³H. Since n-C₁₈ acid is known to be incorporated into the paraffin without loss of carboxyl carbon these results suggest that the condensation of C₁₂ acid with C₁₈ acid is not responsible for n-C₂₉ paraffin synthesis in this tissue. Thus all the experimental evidence thus far obtained strongly suggests that elongation of fatty acids followed by decarboxylation is the most likely pathway for paraffin biosynthesis in leaves.     

Effect of exogenous fatty acids on the growth and production of exopolysaccharides of obligately methylotrophic bacterium Methylophilus quaylei

Accelerating growth and increasing exopolysaccharide production in obligate methylotrophic bacterium Methylophilus quaylei were observed in the presence of C₁₂–C₁₈ fatty acids added to the growth media. Sodium oleate was the best growth factor. Based on data on the composition of the free fatty acids fraction in the cells and the values of the ξ-potential and fluorescence anisotropy of whole cells, we suggested that fatty acids were incorporated in the outer membrane of M. quaylei.