Identification and enumeration of oleic acid and linoleic acid hydrating bacteria in the rumen of sheep and cows

The diversity and population densities of facultative anaerobic bacteria with the capacity to hydrate oleic acid and linoleic acid in the rumen of sheep and dairy cows were determined. The screening of representative colonies, from rumen fluid plated aerobically on a range of agar media, revealed that sheep rumen fluid contained hydration-positive strains of Streptococcus, Staphylococcus, Enterococcus, Lactobacillus and Pediococcus, whereas cow rumen fluid contained hydration-positive strains of Streptococcus, Lactobacillus and Staphylococcus. Mean counts of facultative anaerobic bacteria in sheep and cattle rumen were log10 7.29 and log10 6.40, respectively, and were independent of diet. Approximately 56% of facultative anaerobic bacteria were able to hydrate oleic and/or linoleic acid in anaerobic broth culture. For both sheep and cows, the most numerous hydration-positive isolates were strains of Strep. bovis. The results, which are the first to show that pediococci have the capacity to hydrate unsaturated fatty acids, suggest that lactic acid bacteria are the major unsaturated fatty acid hydrating bacteria in the rumen.     

Oleic acid transformations by selected strains of Sphingobacterium thalpophilum and Bacillus cereus from composted manure

In a survey of 186 randomly selected microbial strains isolated from composted manure, 63 transformed oleic acid into three types of products: hydroxy fatty acid, fatty amide, and less polar oleyl lipid. Selection of oleic acid-transforming microorganisms was enhanced in nutrient agar supplemented with 0.1% (vol/vol) oleic acid at pH 7.2. Most of the 63 diverse isolates elicited inconsistent and poorly reproduced transformations. However, strains 142b (NRRL B-14797) transformed oleic acid to 10-hydroxystearic acid consistently, and strain 229b (NRRL B-14812) produced an octadecenamide. Taxonomic studies indicated that NRRL strain B-14797, possessing 1,3-dihydroxy-2-amino-15-methylhexadecane and sphinganine bases, was closely related to Sphingobacterium thalpophilum, and NRRL B-14812 was identified as Bacillus cereus.     

Anaerobic reduction of ferulic acid to dihydroferulic acid by Wolinella succinogenes from cow rumen

Summary Ferulic acid(FA)-modifying microflora from the rumen of cows were acclimated in an FA-containing medium, in which aromatic compounds (dihydroferulic acid, homovanillic acid, carboxymethylphenol and vanillic acid) and volatile fatty acids (acetic, butyric and isobutyric) were detected by gas chromatography and mass spectrometry. An anaerobic curved bacterium was isolated from the rumen microflora. This bacterium was characterized and identified as Wolinella succinogenes according to the method of Holdeman et al. (1977). It could only reduce FA to dihydroferulic acid in the absence of hydrogen acceptors such as nitrate or fumarate and under strictly anaerobic conditions. FA-reducing ability of the bacterium was inhibited to some extent at FA concentrations greater than 5 mM. The FA was reduced more effectively at pH values of 7.0–7.2 than at 6.8 and the reduction was enhanced by the addition of an Ruminococcus albus' culture supernatant.     

Effects of Long-Chain Fatty Acids on Growth of Rumen Bacteria

The effects of low concentrations of long-chain fatty acids (palmitic, stearic, oleic, and vaccenic) on the growth of seven species (13 strains) of rumen bacteria were investigated. Except for Bacteroides ruminicola and several strains of Butyrivibrio fibrisolvens, bacterial growth was not greatly affected by either palmitic or stearic acids. In contrast, growth of Selenomonas ruminantium, B. ruminicola, and one strain of B. fibrisolvens was stimulated by oleic acid, whereas the cellulolytic species were markedly inhibited by this acid. Vaccenic acid (trans Δ11 18:1) had far less inhibitory effect on the cellulolytic species than oleic acid (cis Δ9 18:1). Inclusion of powdered cellulose in the medium appeared to reverse both inhibitory and stimulatory effects of added fatty acids. However, there was little carry-over effect observed when cells were transferred from a medium with fatty acids to one without. Considerable variation in response to added fatty acids was noted among five strains of B. fibrisolvens. In general, exogenous long-chain fatty acids appear to have little, if any, energy-sparing effect on the growth of rumen bacteria.     

Reliable identification of Prevotella and Butyrivibrio spp. from rumen by fatty acid methyl ester profiles

Data for bacterial identification were provided by culturing anaerobic bacteria under standardized conditions followed by extraction and methylation of cellular long-chain fatty acids and gas chromatographic analysis. The databases of fatty acid methyl ester (FAMEs) profiles for two predominant ruminal genera,Prevotella andButyrivibrio, were created. Major long-chain cellular fatty acids found in the 23 analyzedPrevotella strains were 15:0 (anteiso), 15:0, 15:0 (iso) and 16:0. The strains ofPrevotella could be well identified on species level by the characteristic ratios among major fatty acids and by acids unique fatty for each species. The 45Butyrivibrio strains were grouped into 4 major and 2 minor groups according to FAMEs profiles. The major fatty acids for the bulk of theButyrivibrio strains were 14:0, 15:1, 16:0 and 16:0 (iso). This groups corresponded to those based on 16S rDNA sequences.     

Lipid Metabolism of Rumen Ciliates and Bacteria: II. Uptake of Fatty Acids and Lipid Analysis of Isotrichia intestinalis and Rumen Bacteria with Further Information on Entodinium simplex

The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C¹⁴-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells.     

Factors affecting the formation of 10-hydroxystearic acid from oleic acid by a ruminal strain of Enterococcus faecalis

 A ruminal strain of Enterococcus faecalis was characterised with respect to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydroxy fatty acid was produced after growth had ceased and the carbon source was almost exhausted. Hydroxy fatty acid production was equally rapid whether the inoculum had been grown in the presence of oleic acid or not, and almost complete conversion was achieved when oleic acid was present at a concentration of up to 0.5% (v/v). Incubation under a hydrogen headspace did not result in biohydrogenation of oleic acid. In pH-controlled batch culture the proportion of oleic acid hydrated varied with the pH of incubation, with more hydration at lower pH. Growth was retarded in the presence of 0.1% (v/v) linoleic acid, inhibited by the same concentration of linolenic acid and did not result in the formation of hydrated products from these substrates. If this organism is able to transform oleic acid in the rumen then the only product likely to be formed is 10hydroxystearic acid. Received: 17 July 1995/Received last revision: 24 October 1995/Accepted: 30 October 1995     

Fatty acid and carotenoid composition ofRhodotorula strains

Lipid content and composition of fatty acids with 6–25 carbon atoms were studied on strains of the 13 pink or red yeast species belonging to the genus Rhodotorula. The total amount of lipid represented an average of 13% of the dry weight. The neutral and polar lipid fractions were analyzed separately. For all the strains studied, the major fatty acids in both fractions were oleic, linoleic and palmitic acids, which formed 80% of the total number of fatty acids. A notable amount of arachidonic acid, a precursor of eicosanoid hormones, was found in R. acheniorum, R. aurantiaca and R. bacarum. Depending on the strain, 1–10 carotenoid pigments were detected; β-carotene was always the major carotenoid present. Received: 13 December 1994 / Accepted: 18 May 1995     

Conversion of fatty acids by Bacillus sphaericus-like organisms

Bacillus sphaericus species are mesophilic round-spored organisms that readily utilize fatty acid-based surfactants during growth, but their ability to modify fatty acids is unknown. Among 57 B. sphaericus-like strains tested for fatty acid transformation activity in Wallen fermentation (WF) medium, ten converted oleic acid to a new product determined by gas chromatography – mass spectrometry (GC-MS) to be 10-ketostearic acid (10-KSA). Additionally, a few other strains converted ricinoleic acid and linoleic acid to new products that remain to be characterized. Unlike most microbial hydrations of oleic acid, which produce a mixture of 10-KSA and 10-hydroxystearic acid, the conversion of oleic acid by B. sphaericus strains was unique in that 10-KSA was the sole reaction product. By replacing dextrose with sodium pyruvate in WF and adjusting to pH 6.5, conversion of oleic acid to 10-KSA by strain NRRL NRS-732 was improved from about 11% to more than 60%. Using the defined optimal conditions, the conversion reaction was scaled up in a stirred-batch reactor by using technical-grade oleic acid as substrate. This is the first report on the characterization of fatty acid conversions by B. sphaericus species. Received: 17 December 2001 / Accepted: 17 January 2002     

Antimicrobial activity of fatty acids from fruits of Peucedanum cervaria and P. alsaticum

Plants of the genus Peucedanum have been used in traditional medicine for a long time to treat different diseases including infectious diseases. The hexane fruits extracts of Peucedanum cervaria and P. alsaticum were examined for antimicrobial activity and analyzed for their fatty acid content. Fatty acid composition of oils were analyzed by GC/FID in methyl ester form. Minimal inhibitory concentrations (MICs) of fatty acid fractions against twelve reference bacterial and yeast strains were performed by the twofold serial microdilution broth method. Fourteen fatty acids were identified. Oleic and linoleic acids were found to be dominant. The extracts from both plants examined exhibited inhibitory effects against Gram‐positive strains tested with different MIC values (0.25–2 mg/ml); however, extract from P. alsaticum possessed stronger antibacterial properties and a broader spectrum. The growth of Gram‐negative bacteria and Candida spp. strains was not inhibited even at the highest extract concentration used (MIC>4 mg/ml). Standard fatty acids exhibited inhibitory effects towards all bacterial and yeast strains used in this study; however, the majority of bacteria were more sensitive to linoleic than to oleic acid. These results revealed, for the first time, that hexane extracts obtained from fruits of P. alsaticum and P. cervaria possess moderate in vitro antibacterial activity against Gram‐positive bacteria including staphylococci. Linoleic and oleic acids appear to be the compounds responsible for this effect, and a synergistic antimicrobial effect between these two fatty acids was indicated.     

Adaptation of anaerobically grown Thauera aromatica,Geobacter sulfurreducens and Desulfococcus multivorans to organic solvents on the level of membrane fatty acid composition

The effect of different solvents and pollutants on the cellular fatty acid composition of three bacterial strains: Thauera aromatica, Geobacter sulfurreducens and Desulfococcus multivorans, representatives of diverse predominant anaerobic metabolisms was investigated. As the prevailing adaptive mechanism in cells of T. aromatica and G. sulfurreducens whose cellular fatty acids patterns were dominated by palmitic acid (C16:0) and palmitoleic acid (C16:1cis), the cells reacted by an increase in the degree of saturation of their membrane fatty acids when grown in the presence of sublethal concentrations of the chemicals. Next to palmitic acid C16:0, the fatty acid pattern of D. multivorans was dominated by anteiso-branched fatty acids which are characteristic for several sulfate-reducing bacteria. The cells responded to the solvents with an increase in the ratio of straight-chain saturated (C14:0, C16:0, C18:0) to anteiso-branched fatty acids (C15:0anteiso, C17:0anteiso, C17:1anteisoΔ9cis). The results show that anaerobic bacteria react with similar mechanisms like aerobic bacteria in order to adapt their membrane to toxic organic solvents. The observed adaptive modifications on the level of membrane fatty acid composition can only be carried out with de novo synthesis of the fatty acids which is strictly related to cell growth. As the growth rates of anaerobic bacteria are generally much lower than in the so far investigated aerobic bacteria, this adaptive response needs more time in anaerobic bacteria. This might be one explanation for the previously observed higher sensitivity of anaerobic bacteria when compared with aerobic ones.     

Long-chain fatty acid composition of selected genera of yeasts belonging to the Endomycetales

The cellular long-chain fatty acids of 36 strains representing 18 genera of the Saccharomycetaceae, Endomycetaceae, Metchnikowiaceae, Saccharomycodaceae, Schizosaccharomycetaceae and Dipodascaceae were extracted and analyzed as methyl esters by gas chromatography. On the basis of their fatty acid content the set of strains was divided into 6 groups, coinciding with the above families. The members of the Saccharomycetaceae (group I) had a high percentage of oleic acid while the strains classified under the Endomycetaceae (group II) and Metchnikowiaceae (group III) were characterized by oleic acid and linoleic acid as major fatty acids. The Saccharomycodaceae (group IV) had the highest percentage of palmitoleic acid. The Schizosaccharomycetaceae (group V) had the highest percentage of oleic acid, while the Dipodascaceae (group VI) were characterized by a high percentage of linoleic acid.This article originally appeared in an incorrect form in Antonie van Leeuwenhoek, Vol. 52, No. 1.     

Phototrophic bacterial production of oleic acid in an illuminated upflow anaerobic sludge blanket reactor

Phototrophic bacterial cells in the effluent from a lighted upflow anaerobic sludge blanket reactor supplied with a medium containing 142 mg S (as SO₄ ^2–) l^–1 accumulated a 6.8% w/w oleic acid content in cells and 19 mg cell-bound oleic acid l^–1 in the effluent. Pure cultures of Rhodopseudomonas palustris and Blastochloris sulfoviridis isolated from the effluent also accumulated 5.1 and 6.4% w/w oleic acid contents in cells, respectively. The oleic acid content in the cells recovered from the LUASB reactor effluent was related to the phototrophic bacterial population in the LUASB reactor. The inverse relationship was observed in the LUASB reactor between phototrophic bacterial growth and sulfate concentration in the influent.     

Hydration of linoleic acid by bacteria isolated from ruminants

Two strains of Enterococcus faecalis isolated from the ovine rumen and known to hydrate oleic acid were shown to transform linoleic acid by hydration into two products. The products, identified as 10-hydroxy-12-octadecenoic acid and 13-hydroxy-9-octadecenoic acid, were formed during stationary phase in yields of 13% and 6% respectively. Yields increased to 22% and 14% when culture conditions were optimised. To our knowledge, this is the first report of 13-hydroxy-9-octadecenoic acid production by bacteria. During a search for further linoleic-acid-hydrating bacteria, a strain of Streptococcus bovis isolated from bovine faeces and the ruminal strain S. bovis JB1 were found to hydrate linoleic acid. Both strains formed only one product and the most rapid appearance occurred during exponential growth. The S. bovis product, identified as 13-hydroxy-9-octadecenoic acid, formed in a yield of 28%. This study provides the first information on linoleic acid hydration by ruminal bacteria.     

Hydration of Oleic Acid by Enterococcus gallinarum, Pediococcus acidilactici andLactobacillus sp. Isolated from the Rumen

Three ruminal bacteria found to convert oleic acid to 10-hydroxystearic acid were identified asEnterococcus gallinarum , Pediococcus acidilactici and a lactobacillus physiologically similar toLactobacillus reuteri and L. fermentum. The oleic acid-hydrating properties of the three bacteria were determined and hydration was shown to be predominantly a feature of anaerobic culture with relatively little hydration occurring in aerobic culture. Hydration was highly pH dependent and not related to cell growth. At the optimal hydration pH for each bacterium, hydration yields were 97%, 93% and 76% for the E. gallinarum, P. acidilactici andLactobacillus strains, respectively. The enterococcus and the lactobacillus hydrated oleic acid only after growth ceased whereas the pediococcus hydrated oleic acid during the late logarithmic growth. Hydration was not specific for oleic acid with all three bacteria hydrating the oleic acid homologue palmitoleic acid. None of the bacteria hydrogenated oleic acid to stearic acid. Our results suggest that a capacity to hydrate oleic acid may be a property of many lactic acid bacteria.     

Cellular fatty acid composition of Leuconostoc oenos

The cellular fatty acid composition of 70 lactic acid bacteria was examined by capillary gas chromatography. Fifty-four Leuconostoc oenos strains, including three reference, type strains from the other Leuconostoc spp., nine Pediococcus spp. and two Lactobacillus spp. were studied. Eighteen fatty acids were determined, of which 10 were identified by gas chromatography-mass spectrometry. The relative percentages of the 18 fatty acids of the Leuconostoc strains were analyzed numerically and grouped using the unweighted pair-group method. Results show that four clusters could be defined at r = 0.920, with five strains unassigned. The major fatty acids of the Leuc. oenos strains were found to be palmitic acid (C16:0), palmitoleic acid (C16:1–9), oleic acid (C18: 1–9), vaccenic acid (C18: 1–11), dihyrosterculic acid (C19-cyclopropane-9) and lactobacillic acid (C19-cyclopropane-11). It was mainly on the basis of the amounts of oleic acid and the C19-cyclopropane fatty acids that the strains of Leuc. oenos could be distinguished from each other. This is the first report of the occurrence of dihydrosterculic acid in lactic acid bacteria. For the majority of Leuc. oenos strains, the result obtained with the cellular fatty acid analysis confirmed the phenotypic relationships.     

Production of 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from olive oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Microbial modification of naturally occurring materials is one of the efficient ways to add new values to them. Hydroxylation of free unsaturated fatty acids by microorganism is a good example of those modifications. Among microbial strains studied for that purpose, a new bacterial isolate Pseudomonas aeruginosa PR3 has been well studied to produce several hydroxy fatty acids from different unsaturated fatty acids. Of those hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was efficiently produced from oleic acid by strain PR3. However, it was highly plausible to use vegetable oil containing oleic acid rather than free oleic acid as a substrate for DOD production by strain PR3. In this study, we firstly tried to use olive oil containing high content of oleic acid as a substrate for DOD production. DOD production from olive oil was confirmed by structural determination with GC, TLC, and GC/MS analysis. DOD production yield from olive oil was 53.5%. Several important environmental factors were also tested. Galactose and glutamine were optimal carbon and nitrogen sources, and magnesium ion was critically required for DOD production from olive oil. Results from this study demonstrated that natural vegetable oils containing oleic acid could be used as efficient substrate for the production of DOD by strain PR3.     

Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis

Background  

The original anaerobic unsaturated fatty acid biosynthesis pathway proposed by Goldfine and Bloch was based on in vivo labeling studies in Clostridium butyricum ATCC 6015 (now C. beijerinckii) but to date no dedicated unsaturated fatty acid biosynthetic enzyme has been identified in Clostridia. C. acetobutylicium synthesizes the same species of unsaturated fatty acids as E. coli, but lacks all of the known unsaturated fatty acid synthetic genes identified in E. coli and other bacteria. A possible explanation was that two enzymes of saturated fatty acid synthesis of C. acetobutylicium, FabZ and FabF might also function in the unsaturated arm of the pathway (a FabZ homologue is known to be an unsaturated fatty acid synthetic enzyme in enterococci).     

Analysis of fatty acid composition of anaerobic rumen fungi

The fatty acid (FA) composition of fresh mycelia of anaerobic rumen fungi was determined. The fatty acids methyl esters (FAME) of six strains belonging to four genera (Neocallimastix, Caecomyces, Orpinomyces, Anaeromyces) and one unknown strain were analyzed by gas chromatography. All studied fungi possess the same FAs but differences were found in their relative concentrations. The FA profile of anaerobic fungi comprises carbon chains of length ranging from 12 to 24; the most common fatty acids were stearic (C(18:0)), arachidic (C(20:0)), heneicosanoic (C(21:0)), behenic (C(22:0)), tricosanoic (C(23:0)) and lignoceric (C(24:0)) with relative amount representing >4% of total FA. Significant differences were determined for heptadecanoic, oleic, behenic and tricosanoic acids. Rumen anaerobic fungi can contain very long chain fatty acids; we found unsaturated fatty acids including cis-11-eicosenoic (C(20:1)), cis-11,14-eicosadienoic (C(20:2)), erucic (C(22:1n9)), cis-13,16-docosadienoic (C(22:2)) and nervonic (C(24:1)) acids in very small amounts but their presence seems to be unique for anaerobic fungi.     

Characterisation of hexane-degrading microorganisms in a biofilter by stable isotope-based fatty acid analysis, FISH and cultivation

The hexane-degrading bacterial community of a biofilter was characterised by a combination of stable isotope-based phospholipid fatty acid analyses, fluorescence in situ hybridisation and cultivation. About 70 bacterial strains were isolated from a full-scale biofilter used for treatment of hexane containing waste gas of an oil mill. The isolation approach led to 16 bacterial groups, which were identified as members of the Alpha-, Beta- and Gammaproteobacteria, Actinobacteria and Firmicutes. Three groups showed good growth on hexane as the sole source of carbon. These groups were allocated to the genera Gordonia and Sphingomonas and to the Nevskia-branch of the Gammaproteobacteria. Actively degrading populations in the filter material were characterised by incubation of filter material samples with deuterated hexane and subsequent phospholipid fatty acid analysis. Significant labelling of the fatty acids 16:1 cis10, 18:1 cis9 and 18:0 10methyl affiliated the hexane-degrading activity of the biofilter with the isolates of the genus Gordonia. In vitro growth on hexane and in situ labelling of characteristic fatty acids confirmed the central role of these organisms in the hexane degradation within the full-scale biofilter.