Identification and cultivation of anaerobic, syntrophic long-chain fatty acid-degrading microbes from mesophilic and thermophilic methanogenic sludges

We investigated long-chain fatty acid (LCFA)-degrading anaerobic microbes by enrichment, isolation, and RNA-based stable isotope probing (SIP). Primary enrichment cultures were made with each of four LCFA substrates (palmitate, stearate, oleate, or linoleate, as the sole energy source) at 55 degrees C or 37 degrees C with two sources of anaerobic granular sludge as the inoculum. After several transfers, we obtained seven stable enrichment cultures in which LCFAs were converted to methane. The bacterial populations in these cultures were then subjected to 16S rRNA gene-based cloning, in situ hybridization, and RNA-SIP. In five of seven enrichment cultures, the predominant bacteria were affiliated with the family Syntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylum Firmicutes and the class Deltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the family Syntrophomonadaceae, and strain TOL, affiliated with the phylum Firmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association with Methanospirillum hungatei. Even though strain TOL assimilated [(13)C]palmitate in the original enrichment culture, strain TOL has not shown the ability to degrade LCFAs after isolation. These results suggest that microbes involved in the degradation of LCFAs under methanogenic conditions might not belong only to the family Syntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.     

Neutral fat hydrolysis and long-chain fatty acid oxidation during anaerobic digestion of slaughterhouse wastewater

Neutral fat hydrolysis and long-chain fatty acid (LCFA) oxidation rates were determined during the digestion of slaughterhouse wastewater in anaerobic sequencing batch reactors operated at 25 degrees C. The experimental substrate consisted of filtered slaughterhouse wastewater supplemented with pork fat particles at various average initial sizes (D(in)) ranging from 60 to 450 microm. At the D(in) tested, there was no significant particle size effect on the first-order hydrolysis rate. The neutral fat hydrolysis rate averaged 0.63 +/- 0.07 d(-1). LCFA oxidation rate was modelled using a Monod-type equation. The maximum substrate utilization rate (kmax) and the half-saturation concentration (Ks) averaged 164 +/- 37 mg LCFA/L/d and 35 +/- 31 mg LCFA/L, respectively. Pork fat particle degradation was mainly controlled by LCFA oxidation rate and, to a lesser extent, by neutral fat hydrolysis rate. Hydrolysis pretreatment of fat-containing wastewaters and sludges should not substantially accelerate their anaerobic treatment. At a D(in) of 450 microm, fat particles were found to inhibit methane production during the initial 20 h of digestion. Inhibition of methane production in the early phase of digestion was the only significant effect of fat particle size on anaerobic digestion of pork slaughterhouse wastewater. Soluble COD could not be used to determine the rate of lipid hydrolysis due to LCFA adsorption on the biomass.     

Mineralization of LCFA associated with anaerobic sludge: Kinetics, enhancement of methanogenic activity, and effect of VFA

Long-chain fatty acids (LCFA) associated with anaerobic sludge by mechanisms of precipitation, adsorption, or entrapment can be biodegraded to methane. The mineralization kinetics of biomass-associated LCFA were established according to an inhibition model based on Haldane's enzymatic inhibition kinetics. A value around 1,000 mg COD-LCFA..g VSS(-1) was obtained for the optimal specific LCFA content that allowed the maximal mineralization rate. For sludge with specific LCFA contents of 2,838 +/- 63 and 4,571 +/- 257 mg COD-LCFA..g VSS(-1), the specific methanogenic activities in the presence of acetate, butyrate, and H(2)/CO(2) were significantly enhanced after the mineralization of the biomass-associated LCFA. For sludge with a specific LCFA content near the optimal value defined by the kinetic model, the effect of adding VFA to the medium was studied during the mineralization of the biomass-associated LCFA. Different patterns were obtained for each individual substrate. Acetate and butyrate were preferentially consumed by the consortium, but in the case of propionate no evidence of a sequential consumption pattern could be withdrawn. It was concluded that LCFA do not exert a bactericidal neither a permanent toxic effect toward the anaerobic consortia. A discussion is addressed to the relative roles of a reversible inhibitory effect and a transport limitation effect imposed by the LCFA surrounding the cells.     

Enhancement of methane production from long chain fatty acid based effluents

Two anaerobic sludges previously loaded with oleate and palmitate accumulated 4570+/-257 and 5200+/-9 mgCOD-LCFAgVSS(-1), respectively. These sludges were incubated in batch assays and methane production was recorded after addition of 100-900 mg L(-1) of oleate and palmitate, respectively. The batch assays were conducted before and after allowing the depletion of the biomass-associated LCFA. The presence of biomass-associated LCFA decreased the capacity of both sludges to convert the added LCFA to methane. After the degradation of biomass-associated LCFA, the lag phases observed before the onset of methane production were significantly reduced, evidencing an increase in the tolerance of the acetotrophic methanogens to the presence of LCFA. In another experiment, three recurrent pulses were performed with a real dairy wastewater containing 3.6 gCOD L(-1), from which 53% was fat. Methane yields of 0.45+/-0.01, 0.88+/-0.02 and 1.29+/-0.08 gCOD-CH(4) gCOD(fed)(-1) were achieved in the first, second and third pulses, respectively, evidencing an increasing capacity of the sludge to convert substrate accumulated in previous additions.     

Effects of emulsified octadecanic acids on gas production and cellulolysis by the rumen anaerobic fungus, Piromyces communis M014

Responses of the rumen anaerobic fungus, Piromyces communis M014, to octadecanic long-chain fatty acids (LCFAs) were evaluated by measuring total and hydrogen gas productions, filter paper (FP) cellulose degradation and polysaccharidase enzyme activities. Octadecanic acids (stearic acid, C(18:0); oleic acid, C(18:1); linoleic acid, C(18:2) and linolenic acid, C(18:3)) were emulsified by ultrasonication under anaerobic conditions, and added to the medium at the level of 0.001%. When P. communis M014 was grown in culture with stearic and oleic acids, the cumulative gas production, FP cellulose digestion and enzyme activities were significantly (p<0.05) increased in the early incubation times relative to those for the control. However, the addition of linolenic acid inhibited all of the investigated parameters, including cellulose degradation, enzyme activities and gas production, up to 168h incubation. These results indicated that stearic and oleic acids tended to have stimulatory effects on fungal cellulolysis, whereas linolenic acid caused a significant (p<0.05) inhibitory effect on cellulolysis by the rumen fungus. The fungus, P. communis M014, can biohydrogenate C(18) unsaturated fatty acids to escape from their toxic effects. Therefore, in this study, the results indicated that the more highly the added C(18) LCFA to the fungal culture was unsaturated, the higher the inhibition of gas production and cellulase enzyme activity was.     

Localization of adipocyte long-chain fatty acyl-CoA synthetase at the plasma membrane

Long-chain fatty acyl-CoA synthetase (FACS) catalyzes esterification of long-chain fatty acids (LCFAs) with coenzyme A (CoA), the first step in fatty acid metabolism. FACS has been shown to play a role in LCFA import into bacteria and implicated to function in mammalian cell LCFA import. In the present study, we demonstrate that FACS overexpression in fibroblasts increases LCFA uptake, and overexpression of both FACS and the fatty acid transport protein (FATP) have synergistic effects on LCFA uptake. To explore how FACS contributes to LCFA import, we examined the subcellular location of this enzyme in 3T3-L1 adipocytes which natively express this protein and which efficiently take up LCFAs. We demonstrate for the first time that FACS is an integral membrane protein. Subcellular fractionation of adipocytes by differential density centrifugation reveals immunoreactive and enzymatically active FACS in several membrane fractions, including the plasma membrane. Immunofluorescence studies on adipocyte plasma membrane lawns confirm that FACS resides at the plasma membrane of adipocytes, where it co-distributes with FATP. Taken together, our data support a model in which imported LCFAs are immediately esterified at the plasma membrane upon uptake, and in which FATP and FACS function coordinately to facilitate LCFA movement across the plasma membrane of mammalian cells.     

Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.

Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.     

Toluene bioconversion to p-hydroxybenzoate by fed-batch cultures of recombinant Pseudomonas putida.

A microbial oxidation process for the production of p-hydroxybenzoate (HBA) from toluene is reported. The oxidation reaction was studied in fed-batch fermentations using a recombinant Pseudomonas putida grown on glutamate as the sole carbon and energy source with salicylate and IPTG induction of tmoABCDE, and pchCF and phbz pathway genes, respectively. An average volumetric HBA productivity of 13.4 mg HBA x L(-1) x h(-1) was obtained under rapid growth conditions (glutamate excess), giving an HBA titer of 132 mg x L(-1) after 9.8 h of fermentation. This corresponded to an average specific HBA productivity of 7.2 microg HBA (mg total protein)(-1) x h(-1). In contrast, maximum HBA titers of 35 mg HBA x L(-1) were achieved in 27 h in comparative studies employing glutumate limited fed-batch cultures. A specific productivity of 4.1 microg HBA (mg total protein)(-1) x h(-1) and volumetric productivity of 1.3 mg HBA x L(-1) x h(-1) were calculated for the growth-rate restricted cultures. The differences in HBA production between the two cultures could be correlated to the levels of specific toluene-4-monooxygenase (T4MO) polypeptides. T4MO catalyzes the rate-limiting step in the pathway. Using experimental data, the half-life value of TmoA was calculated to be approximately 28 h. Assuming linear, monomolecular decay of TmoA, a specific degradation constant of 0.025 x h(-1) was calculated, which placed the stability of recombinant TmoA in the range of relatively stable proteins, even in the absence of co-expression of tmoF, the terminal oxidoreductase subunit of T4MO.     

The long-chain fatty acid sensor, PsrA, modulates the expression of rpoS and the type III secretion exsCEBA operon in Pseudomonas aeruginosa

The Pseudomonas aeruginosa PsrA autorepressor has dual roles as a repressor of the fadBA5 β-oxidation operon and an activator of the stationary-phase sigma factor rpoS and exsCEBA operon of the type III secretion system (TTSS). Previously, we demonstrated that the repression of the fadBA5 operon by PsrA is relieved by long-chain fatty acids (LCFAs). However, the signal affecting the activation of rpoS and exsC via PsrA is unknown. In this study, microarray and gene fusion data suggested that LCFA (e.g. oleate) affected the expression of rpoS and exsC . DNA binding studies confirmed that PsrA binds to the rpoS and exsC promoter regions. This binding was inhibited by LCFA, indicating that LCFA directly affects the activation of these two genes through PsrA. LCFA decreased rpoS and exsC expression, resulting in increased N -(butyryl)- l -homoserine-lactone quorum sensing signal and decreased ExoS/T production respectively. Based on the crystal structure of PsrA, site-directed mutagenesis of amino acid residues, within the hydrophobic channel thought to accommodate LCFA, created two LCFA-non-responsive PsrA mutants. The binding and activation of rpoS and exsC by these PsrA mutants was no longer inhibited by LCFA. These data support a mechanistic model where LCFAs influence PsrA regulation to control LCFA metabolism and some virulence genes in P. aeruginosa .     

Impact of Chlorofluorocarbon 113 on Chlorinated Ethene Biodegradation

The inhibition of tetrachloroethene (PCE) degradation in anaerobic, ethanol-fed PCE-enrichment cultures by chlorofluorocarbon 113 (CFC113) was a function of the initial CFC113 concentration. Typically, aqueous CFC113 concentrations up to 1 mg/L slowed, but did not stop PCE-degradation, but cis-1,2-dichloroethene (cDCE) degradation was inhibited by 0.2 mg/L CFC113. In some cultures, however, PCE degradation was stopped by as little as 0.15 mg/L CFC113. CFC113 also slowed the consumption of hydrogen and the concurrent methane production. CFC113 slowly degraded in PCE-enrichment cultures to hydrochlorofluorocarbon 123a (HCFC123a). Chlorotrifluoroethene was also detected. Although relatively non-toxic, CFC113 may nevertheless pose remediation challenges when present at sites that also contain PCE.     

Impact of Chlorofluorocarbon 113 on Chlorinated Ethene Biodegradation

The inhibition of tetrachloroethene (PCE) degradation in anaerobic, ethanol-fed PCE-enrichment cultures by chlorofluorocarbon 113 (CFC113) was a function of the initial CFC113 concentration. Typically, aqueous CFC113 concentrations up to 1 mg/L slowed, but did not stop PCE-degradation, but cis-1,2-dichloroethene (cDCE) degradation was inhibited by 0.2 mg/L CFC113. In some cultures, however, PCE degradation was stopped by as little as 0.15 mg/L CFC113. CFC113 also slowed the consumption of hydrogen and the concurrent methane production. CFC113 slowly degraded in PCE-enrichment cultures to hydrochlorofluorocarbon 123a (HCFC123a). Chlorotrifluoroethene was also detected. Although relatively non-toxic, CFC113 may nevertheless pose remediation challenges when present at sites that also contain PCE.     

Hydrogen formation by cyanobacteria cultures selected for nitrogen fixation

Seventy-one cyanobacteria containing cultures were enriched from various soil and water locations either under aerobic and/or anaerobic conditions on agar medium selective for nitrogen fixation. Kept under argon containing 1% CO₂ for 24 and 48 h most of these cultures evolved hydrogen at very variable rates up to 116 l per mg chlorophyll and hour as a mean value over a time period of 24h. Several samples evolved hydrogen more efficiently compared with known hydrogen producing pure strains from culture collections. Thirty-one of the investigated cultures showed a hydrogen formation higher than 10 l per mg chlorophyll and hour measured over 24 or 48 h. Among these all the morphological forms of cyanobacteria i.e. unicellular and filamentous with or without heterocysts are found. Hence, selecting for nitrogen fixing cyanobacteria seems to be a practical method to find efficient hydrogen producers.     

Real-time quantification of fatty acid uptake using a novel fluorescence assay

Uptake of nonesterified long-chain fatty acids (LCFAs) into many cell types and organs such as liver, heart, intestine, and skeletal muscle occurs primarily through a saturable, protein-mediated mechanism. Membrane proteins that increase the uptake of LCFAs, such as FAT/CD36 and fatty acid transport proteins, represent significant therapeutic targets for the treatment of metabolic disorders, including type 2 diabetes. However, currently available methods for the quantification of LCFA uptake neither allow for real-time measurements of uptake kinetics nor are ideally suited for the development of LCFA uptake inhibitors in high-throughput screens. To address both problems, we developed a LCFA uptake assay using a fluorescently labeled fatty acid and a nontoxic cell-impermeable quenching agent that allows fatty acid transport to be measured in real time using fluorescence plate readers or standard fluorescence microscopy. With this assay, we faithfully reproduced known differentiation- and hormone-induced changes in LCFA uptake by 3T3-L1 cells and determined LCFA uptake kinetics with previously unobtainable temporal resolution. Applications of this novel assay should facilitate new insights into the biology of fatty acid uptake and provide new means for obesity-related drug discovery.     

Sulfo-N-succinimidyl esters of long chain fatty acids specifically inhibit fatty acid translocase (FAT/CD36)-mediated cellular fatty acid uptake

Sulfo-N-succinimidyl esters of LCFAs are a powerful tool to investigate the functional significance of plasmalemmal proteins in the LCFA uptake process. This notion is based on the following observations. First, sulfo-N-succinimidyl oleate (SSO) was found to inhibit the bulk of LCFA uptake into various cell types, i.e. rat adipocytes, type II pneumocytes and cardiac myocytes. Second, using cardiac giant membrane vesicles, in which LCFA uptake can be investigated in the absence of mitochondrial -oxidation, SSO retained the ability to largely inhibit LCFA uptake, indicating that inhibition of LCFA transsarcolemmal transport is its primary action. Third, SSO has no inhibitory effect on glucose and octanoate uptake into giant membrane vesicles derived from heart and skeletal muscle, indicating that its action is specific for LCFA uptake. Finally, SSO specifically binds to the 88 kDa plasmalemmal fatty acid transporter FAT, a rat homologue of human CD36, resulting in an arrest of the transport function of this protein.In addition to its inhibitory action at the plasma membrane level, evidence is presented for the lack of a direct inhibitory effect on subsequent LCFA metabolism. First, the relative contribution of oxidation and esterification to LCFA uptake is not altered in the presence of SSO. Second, isoproterenol-mediated channeling of LCFAs into oxidative pathways is not affected by sulfo-N-succinimidyl palmitate (SSP). As an example of its application we used SSP to study the role of FAT/CD36 in contraction- and insulin-stimulated LCFA uptake by cardiac myocytes , showing that this transporter is a primary site of regulation of cellular LCFA utilization.     

Defect in human myocardial long-chain fatty acid uptake is caused by FAT/CD36 mutations

Because of the importance of long-chain fatty acids (LCFAs) as a myocardial energy substrate, myocardial LCFA metabolism has been of particular interest for the understanding of cardiac pathophysiology. Recently, by using radiolabeled LCFA analogues, myocardial LCFA metabolism has been clinically evaluated, which revealed a total defect of myocardial LCFA accumulation in a small number of subjects. The mechanism for the cellular LCFA uptake process is still disputable, but recent results suggest that fatty acid translocase (FAT)/CD36 is a transporter in the heart. In the present study, we analyzed mutations and protein expression of the FAT/CD36 gene in 47 patients who showed total lack of the accumulation of a radiolabeled LCFA analogue in the heart. All the patients carried two mutations in the FAT/CD36 gene, and expression of the FAT/CD36 protein was not detected on either platelet or monocyte membranes. Our results showed the link between mutations of the FAT/CD36 gene and a defect in the accumulation of LCFAs in the human heart.     

过量表达异柠檬酸裂解酶对ldh-1谷氨酸棒状杆菌产丁二酸的影响

谷氨酸棒状杆菌SA001是缺失了乳酸脱氢酶基因 (ldhA) 的菌株。为了增加厌氧条件下经异柠檬酸到丁二酸的代谢通量,以提高丁二酸的产量。将来自大肠杆菌Escherichia coli K12的异柠檬酸裂解酶基因导入谷氨酸棒状杆菌SA001 (SA001/pXMJ19-aceA) 中。该菌经0.8 mmol/L的IPTG有氧诱导12 h后,转入厌氧发酵16 h,丁二酸的产量为10.38 g/L,丁二酸的生产强度为0.83 g/(L·h)。与出发菌株比较,异柠檬酸裂解酶的酶活提高了5.8倍,丁二酸的产量提高了48%。结果表明过量表达异柠檬酸裂解酶可以增加由乙醛酸途径流向丁二酸的代谢流。     

Regulation of uncoupling protein 2 expression by long-chain fatty acids and hormones in bovine mammary epithelial cells

Although mammary epithelial cells are known to synthesize and accumulate triacylglycerol (TAG) in order to produce milk lipid in the cytosol, lipid and energy metabolism is still not fully understood. In this study, we assessed the effects of long-chain fatty acid (LCFA) on the accumulation of cytosolic TAG and uncoupling protein (UCP) 2 in cloned bovine mammary epithelial cells (bMEC). LCFAs significantly raised the expression of UCP2 mRNA and the accumulation of TAG. We observed the rapid elevation in UCP2 shown at 6 h after LCFA treatment. Insulin (5-50 ng/ml) or dexamethasone (500 nM) significantly suppressed the expression of UCP2 mRNA. These results suggest that UCP2 play an important role of lipid and energy metabolism in mammary epithelial cells.     

Loranthus acaciae: Alternative medicine for β-lactamase producer and methicillin-resistant Staphylococcus aureus

Recently, we reported high antibacterial efficiency of Loranthus acaciae (LA) against different standard strains of bacteria including Methicillin-Resistant Staphylococcus aureus (MRSA). Therefore, this study aimed to confirm the effectiveness of LA against clinically isolated Staphylococcus aureus (SA) including β-lactamase producer (Blac) and MRSA. Forty-eight SA isolates collected from various clinical samples were used in this study. Antibiotics susceptibility profile was determined for twenty different antibiotics using automated Microscan Walkaway 96 Plus system as recommended by Clinical and Laboratory Standards Institute (CLSI) guidelines. This system also identified β-lactamase producers and MRSA. In the meantime, LA ethanolic extract was fractionated using liquid–liquid fraction method to hexane, dichloromethane DCM and methanol 80% fractions. Antimicrobial activities of LA extract and fraction were performed with agar well diffusion method for all SA isolates, MIC and MBC were also recorded. Phytochemical screening for various phyto-constituent classes of LA ethanolic extract was determined. Out of 48 SA isolates, Cefoxitin-positive MRSA represent 31 (64.6%), Blac 17 (35.4%), and 41 (85.4%) were multidrug-resistant SA, which was resistant at least to one antibiotic from three different categories. All isolates were resistant to ampicillin and penicillin. Antimicrobial activities of LA extract and fractions revealed that ethanol extract was active against all isolated SA with inhibition zone ranged from 33 ± 2.00 to 25 ± 3.05 mm. While DCM exhibited the largest inhibition zone range from 37 ± 3.00 to 33 ± 2.00 mm. This study is first of its kind conforming the high antibacterial activity of LA against SA isolated from a different source of infection. The study concluded that LA extract and fractions are active and give positive result for all isolated SA. Therefore, suitable pharmacological formulation of LA extract as a promising antibacterial agent for the treatment of SA infection should be given extreme priority.