Branched chain amino acids as source of specific branched chain volatile fatty acids during the fermentation process of fish sauce

Summary.  The source of the formation of branched chain volatile fatty acids (VFA) in fish sauce was investigated. Certain branched VFA were derived from the degradation of specific amino acids as iso-butyric acid from valine and iso-valeric acid from leucine. Short and long straight chain VFA were significantly higher in the linoleic acid added sample than in the control but did not significantly bring changes to the branched chain VFA. It is suggested that straight chain VFA developed from fish fats. Alanine and isoleucine did not have a clear influence on the production of volatile fatty acids. Received November 23, 2001 Accepted June 20, 2002 Published online December 18, 2002 RID="*" ID="*" Part of this paper was presented in the 7th International Congress on Amino Acids and Proteins in Vienna, Austria from August 6–10, 2001. Authors' address: Norlita G. Sanceda, Ph.D., Institute of Environmental Science for Human Life, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112, Japan, Fax: + 81-3-5978-5805, E-mail: lita@cc.ocha.ac.jp     

In vivo production and molar percentages of volatile fatty acids in the rumen: a quantitative review by an empirical approach

Despite their major contribution to the energy supply of ruminants, the production of volatile fatty acids (VFA) in the rumen is still poorly predicted by rumen models. We have developed an empirical approach, based on the interpretation of large bibliographic databases gathering published in vivo measurements of ruminal VFA production rate (PR), rates of duodenal and faecal digestion and molar percentages of VFA in the rumen. These databases, covering a wide range of intake levels and dietary composition, were studied by meta-analysis using within-experiment models. We established models to quantify response laws of total VFA-PR and individual VFA molar percentages in the rumen to variations in intake level and dietary composition. The rumen fermentable organic matter (RfOM) intake, estimated from detailed knowledge of the chemical composition of diets according to INRA Feed Tables, appears as an accurate explanatory variable of measured total VFA-PR, with an average increment of 8.03 ± 0.64 mol total VFA/kg RfOM intake. Similar results were obtained when total VFA-PR was estimated from measured apparent RfOM (total VFA-PR/RfOM averaging 8.3 ± 1.2 mol/kg). The VFA molar percentages were related to dry matter intake and measured digestible organic matter (OM), digestible NDF and rumen starch digestibility, with root mean square error of 1.23, 1.45, 0.88 and 0.41 mol/100 mol total VFA for acetate, propionate, butyrate and minor VFA, respectively, with no effect of pH on the residuals. Stoichiometry coefficients were calculated from the slopes of the relationships between individual VFA production (estimated from measured apparent RfOM and individual VFA molar percentages) and measured fermented fractions. Coefficients averaged, respectively, 66, 17, 14 and 3 mol/100 mol for NDF; 41, 44, 12 and 4 mol/100 mol for starch; and 46, 35, 13 and 6 mol/100 mol for crude protein. Their use to predict VFA molar percentages appear relevant for most dietary conditions, that is, when the digested NDF/digested OM ratio exceeded 0.12. This study provides a quantitative review on VFA yield in the rumen. It contributes to the development of feed evaluation systems based on nutrient fluxes.     

Recovery of volatile fatty acids from biological materials for direct analysis by gas chromatography

A simple and efficient methodology for preparing aqueous extracts of volatile fatty acids from biological materials, for direct analysis by gas chromatography is described. Peak areas and responses relative to n-butyric acid were used to calculate concentrations of the individual acids. An example is given for analysis of the volatile fatty acids found in the blood of the lugworm Aerinocola marina.     

Effect of volatile fatty acids on Shigella dysenteriae during anaerobic digestion of human night soil

Thein vitro toxic effect of different volatile fatty acids (VFA) on Shigella dysenteriae was studied in pure culture. Volatile fatty acids viz., acetate, propionate, butyrate, valerate, caproate and heptanoate, exerted pH dependent toxic effect on the pathogen, with minimum inhibitory concentration in the range of 10–3000 mg l⁻¹. The effect of high levels of VFA on S. dysenteriae was studied during anaerobic digestion of human night soil in an experimental digester with VFA level ≅ 9000 mg l⁻¹ and pH ≅ 6.5. Another digester, with VFA level ≅ 700 mg l⁻¹ and pH 7.4, served as the control. In the experimental digester, S. dysenteriae was completely eliminated within 18 days. In the control digester, a four-log reduction in pathogen count was achieved however the pathogen was not completely eliminated. T ₉₀ values for the experimental and control digesters were 2.2 and 3.7 days respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Conversion of volatile fatty acids into polyhydroxyalkanoate by Ralstonia eutropha

Aims:  The aims of this study were to optimize condensed corn solubles (CCS) as a medium for growth of Ralstonia eutropha and to determine the effects of individual volatile fatty acids (VFAs) on polyhydroxyalkanoate (PHA) production .
Methods and Results:  A CCS medium of concentration 240 g l⁻¹ with a carbon : nitrogen ratio of 50 : 1 was developed as the optimal medium. Cultures were grown in 1-l aerated flasks at 250 rev min⁻¹ at 30°C for 120 h. Comparable growth rates were observed in CCS vs a defined medium. At 48 h, VFAs were fed individually at different levels. Optimal levels of all the acids were determined to maximize PHA production. An overall comparison of the VFAs indicated that butyric and propionic acids provided the best results.
Conclusion:  An optimized CCS medium supported growth of R. eutropha . Butyric and propionic acids were the most efficient carbon sources to maximize PHA production when added at the 5 g l⁻¹ level.
Significance and Impact of the Study:  The study shows that a byproduct of ethanol industry can be effectively used as a low cost medium for PHA production, thus partly reducing the cost of commercialization of biopolymers.     

Limitations of thermophilic anaerobic wastewater treatment and the consequences for process design

Thermophilic anaerobic digestion offers an attractive alternative for the treatment of medium- and high-strength wastewaters. However, literature reports reveal that thermophilic wastewater treatment systems are often more sensitive to environmental changes than the well-defined high-rate reactors at the mesophilic temperature range. Also, in many cases a poorer effluent quality is experienced while the carry over of suspended solids in the effluent is relatively high. In this paper recent achievements are discussed regarding the process stability of thermophilic anaerobic wastewater treatment systems. Laboratory experiments reveal a relatively low sensitivity to temperature changes if high-rate reactors with immobilized biomass are used. Other results show that if a staged process is applied, thermophilic reactors can be operated for prolonged periods of time under extreme loading conditions (80–100 kg chemical oxygen demand.m^-3.day^-1), while the concentrations of volatile fatty acids in the effluent remain at a low level.     

Concurrent nitrite oxidation and aerobic denitrification in activated sludge exposed to volatile fatty acids

The goal of this research was to investigate the simultaneous occurrence of nitrification and denitrification by activated sludge exposed to volatile fatty acids (VFAs) during aerobic wastewater treatment using a single-stage reactor. A mixture of VFAs was spiked directly into a continuous-stirred tank reactor (CSTR) to assess subsequent impacts on nitrite removal, nitrate formation, CO(2) fixation, total bacterial density, and dominant nitrite oxidizing bacteria (NOB) concentration (i.e., Nitrospira). The activity of the periplasmic nitrate reductase (NAP) enzyme and the presence of nap gene were also measured. A rapid decrease in the nitrate formation rate (>70% reduction) was measured for activated sludge exposed to VFAs; however, the nitrite removal rate was not reduced. The total bacterial density and Nitrospira concentration remained essentially constant; therefore, the reduction in nitrate formation rate was likely not due to heterotrophic uptake of nitrogen or to a decrease in the dominant NOB population. Additionally, VFA exposure did not impact microbial CO(2) fixation efficiency. The activity of NAP enzyme increased in the presence of VFAs suggesting that nitrate produced as a consequence of nitrite oxidation was likely further reduced to gaseous denitrification products via catalysis by NAP. Little, if any, nitrogen was discharged in the aqueous effluent of the CSTR after exposure to VFAs demonstrating that activated sludge treatment yielded compounds other than those typically produced solely by nitrification.     

Biohydrogenation of C20 polyunsaturated fatty acids by anaerobic bacteria

The PUFAs include many bioactive lipids. The microbial metabolism of C₁₈ PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C₂₀ PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C₂₀ PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C₂₀ PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C₁₈ and C₂₀ PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.     

Volatile fatty acids and anaerobic fermentation in temperate piscivorous and omnivorous freshwater fish

Endproducts of anaerobic fermentation, volatile fatty acids (VFAs), occur in the intestines of fish but no direct comparisons exist between VFA levels in fish with differing feeding ecologies during different seasons. We measured intestinal concentrations of six types of VFA in the upper and lower intestines of two freshwater omnivores ( Cyprinus carpioi and Dorosoma cepedianum ), and one piscivore ( Micropterus salmoides ) during the spring, summer and autumn. Acetate occurred in all species, and was highest in M. salmoides . In all species, concentrations were similar between upper and lower guts and higher during the summer. All three species contained anerobic bacteria and C. carpio and D. cepedianum contained cellulolytic types. Scanning electron microscopy revealed extensive colonization and suggested microbial breakdown of digesta in M. salmoides . In radio-tracer experiments, C. carpio dosed orally with [2–¹⁴C] acetate contained label in liver, muscle and blood tissues. Amounts of intestinal VFA did not appear to increase in species with refractile diets, and low VFA in D. cepedianum suggests fermentation plays a minimal role in the nutrition of this species. Low levels of intestinal VFA during cool seasons are consistent with the hypothesis that temperature limits fermentation in these Species.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Specificity effects in the biosynthesis of fatty acids in Bacillus acidocaldarius

Addition to Bacillus acidocaldarius of acids which can act as primers for fatty acid synthesis promote the synthesis of corresponding fatty acids competitively. The effective acids are n?C₅ to -?₇ (not C₄ or C₈), iso- and anteiso-C, and ?C, (not C4), and a range of cyclic acids from cyclobutylacetic and cyclopentanecarboxylic to cycloheptylacetic. New non-natural ω-cyclobutyl-, ω-cyclopentyl-, and ω-cycloheptyl-fatty acids are obtainable. The range of acceptable primers and the range of fatty acids produced therefrom indicate, respectively, the substrate specificities of the transacylase which introduces acyl species into fatty acids synthesis and the one which removes them. The specificity of the primer transacylase may be similar to that in some rumen anaerobes.     

Effects of branched-chain volatile fatty acids on lactation performance and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows

Branched-chain volatile fatty acids (BCVFA) supplements could promote lactation performance and milk quality by improving ruminal fermentation and milk fatty acid synthesis. This study was conducted to evaluate the effects of BCVFA supplementation on milk performance, ruminal fermentation, nutrient digestibility and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows. A total of 36 multiparous Chinese Holstein cows averaging 606±4.7 kg of BW, 65±5.2 day in milk (DIM) with daily milk production of 30.6±0.72 kg were assigned to one of four groups blocked by lactation number, milk yield and DIM. The treatments were control, low-BCVFA (LBCVFA), medium-BCVFA (MBCVFA) and high-BCVFA (HBCVFA) with 0, 30, 60 and 90 g BCVFA per cow per day, respectively. Experimental periods were 105 days with 15 days of adaptation and 90 days of data collection. Dry matter (DM) intake tended to increase, but BW changes were similar among treatments. Yields of actual milk, 4% fat corrected milk, milk fat and true protein linearly increased, but feed conversion ratio (FCR) linearly decreased with increasing BCVFA supplementation. Milk fat content linearly increased, but true protein content tended to increase. Contents of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0 and C15:0 fatty acids in milk fat linearly increased, whereas other fatty acids were not affected with increasing BCVFA supplementation. Ruminal pH, ammonia N concentration and propionate molar proportion linearly decreased, but total VFA production and molar proportions of acetate and butyrate linearly increased with increasing BCVFA supplementation. Consequently, acetate to propionate ratios linearly increased. Digestibilities of DM, organic matter, CP, NDF and ADF also linearly increased. In addition, mRNA expressions of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding factor 1 and fatty acid-binding protein 3 linearly increased, mRNA expressions of acetyl-coenzyme A carboxylase-α, fatty acid synthase and stearoyl-CoA desaturase quadratically increased. However, lipoprotein lipase mRNA expression was not affected by treatments. The results indicated that lactation performance and milk fat synthesis increased with BCVFA supplementation by improving ruminal fermentation, nutrient digestibility and mRNA expressions of genes related to milk fat synthesis.     

Utilization of volatile fatty acids from the anaerobic digestion liquor of sewage sludge for 5-aminolevulinic acid production by photosynthetic bacteria

Using volatile fatty acids (VFA) from the anaerobic digestion liquor of sewage sludge, up to 9.2 mm 5-aminolevulinic acid (ALA) could be produced by Rhodobacter sphaeroides under anaerobic-light (5 kLux) conditions with repeated addition of levulinic acid (LA) and glycine and using a large inoculum (approx. 2 g/l of cells, initially from glutamate/malate medium). As the VFA medium also contained organic nitrogen sources such as glutamic acid, the cells were later grown up to about 2 g/l in the VFA medium instead of the glutamate/malate medium. ALA production was then again promoted by adding LA and glycine. Using this improved method, up to 9.3 mm ALA was produced by feeding propionate and acetate together with LA and glycine, indicating that VFA medium formed from sewage sludge could be useful for ALA production.     

Betaine increases net portal absorption of volatile fatty acids in Iberian pigs

Betaine is an osmolyte with the potential to increase volatile fatty acids (VFAs) production and hence improve intestinal health.The present study investigated how betaine affects portal and arterial concentrations and net portal absorption (NPA) of VFA in growing Iberian pigs. Eight 30 kg BW Iberian growing barrows with indwelling catheters in portal vein, ileal vein and carotid artery were randomly assigned to a control diet or a diet supplemented with 0.5% betaine. Para-aminohippuric acid was infused into the ileal vein as a marker to determine portal blood flow using the dilution method. Blood samples were simultaneously taken from the carotid artery and portal vein at −60, 60, 120, 180, 240, 300 and 360 min after feeding 1 200 g of the diet. The NPA of VFA (acetate, propionate, butyrate, valerate, isobutyrate and caproate) was determined by multiplying the porto-arterial plasma concentration differences by portal plasma flow. Betaine increased NPA of acetate (1.44 fold; P < 0.001) and total VFA (0.55 fold; P < 0.001) while decreased NPA of propionate (−0.38 fold; P < 0.05) and valerate (−1.46 fold; P < 0.05) compared with control pigs. Estimated heat production potentially derived from NPA of VFA accounted for 0.20–0.27 of metabolizable energy for maintenance. Acetate and propionate accounted for most of the total VFA estimated heat production (0.83–0.89). Regarding bacterial communities, betaine apparently did not change the DNA abundance of fecal total bacteria, Lactobacillus, Bifidobacterium, Enterobacteriaceae, Bacteroides and the Clostridium clusters I, IV and XIV. In conclusion, betaine increased portal appearance and NPA of VFA, contributing to cover maintenance energy requirements.     

Effects of age and mannanoligosaccharides supplementation on production of volatile fatty acids in the caecum of rabbits

This trial studied the effect of including mannanoligosaccharides (MOS, Bio-Mos^®, Alltech Inc., USA) in the diet on the caecal volatile fatty acids (VFA) and pH of rabbits from 34 to 90 days of age. Three experimental diets were compared: control diet, zinc bacitracin (ZnBac) diet (control diet with 0.1 g ZnBac/kg feed) and MOS diet (control diet with 2.0 g MOS/kg feed). Rabbits were slaughtered at 34, 48, 69 and 90 days of age and caecal contents were collected and analyzed for dry matter, pH and VFA concentration. The empty caecum and caecal contents weights relative to live weight were also determined.Age affected (P<0.0001) VFA concentration and pH values in the caecum. The pH decreased with age whereas VFA concentration increased. Rabbits fed MOS had higher (P<0.05) VFA and tended (P=0.098) to had lower pH in the caecum than rabbits fed ZnBac and control diets. Acetic, propionic and butyric acids concentrations in the caecum increased with MOS but its molar proportions were similar among diets. Diet had no effect on empty caecum weight and caecal contents weight and dry matter concentration. There was no interaction effect between diet and age. From 34 to 90 days of age, VFA production was higher (P<0.05) in the caecum of rabbits fed MOS than in those fed ZnBac diet and control diet.The addition of MOS to the diet increased the VFA concentration in the caecum of growing rabbits from 34 to 90 days of age.     

Signature fatty acids in the polar lipids of acid-producing Thiobacillus spp.: Methoxy, cyclopropyl, alpha-hydroxy-cyclopropyl and branched and normal monoenoic fatty acids

Abstract The polar lipids of 5 species of Thiobacillus were extracted and purified. An analysis of the fatty acid composition of the polar lipids documented the presence of methoxy, cyclopropyl, monounsaturated and hydroxycyclopropyl fatty acids of sufficiently unusual structure to serve as 'signatures' for the presence of these organisms in environmental samples. The structures of the unusual fatty acids of the polar lipids were confirmed by mass spectrometry (MS) after isolation by capillary gas chromatography (GC).     

Biochemistry of radioiodinated free fatty acids

Summary Radioiodinated free fatty acids have been developed to study myocardial metabolism non-invasively in man. In the present study the distribution of radiolabeled lipids in the myocardium and in arterial and coronary sinus blood was evaluated following injection of three commonly used iodinated fatty acids in fasted (n = 5) and lactate loaded (n = 3) dogs. Five minutes after simultaneous i.v. injection of radioiodinated 17-I-heptadecanoic acid (IHDA),15-(p-I-phenyl) pentadecanoic acid (IPPA) and 15-(p-I-phenyl)-3,3-dimethylpentadecanoic acid (DMIPPA) a biopsy specimen and samples of arterial and coronary sinus blood were taken. After extraction and TLC the relative distribution of radioactivity in the aqueous phase (containing the oxidation products), pellet and organic phase was calculated. The organic phase was further divided into phospholipids, diglycerides, free fatty acids, triglycerides and cholesterolesters. Seventy two percent of IHDA was oxidized, 36% of IPPA and 7% of DMIPPA. The organic phase consisted primarily of triglycerides and phospholipids. The ratios of triglycerides to phospholipids were about the same for IHDA, IPPA and DMIPPA (0.58, 0.65 and 0.50, respectively). Free IHDA in tissue samples was low (4%) and elevated for IPPA and DMIPPA, (17% and 37%). During lactate loading triglycerides were higher for all three fatty acids. For IHDA and IPPA this increase was paralleled by a decrease in the aqueous phase, in case of DMIPPA the aqueous phase remained the same. Five minutes after injection most of the organic phase of both arterial and coronary sinus blood consisted of the injected fatty acids, the aqueous phase contained oxidation products. There were only minor differences during lactate loading. During the evaluation of scintigraphic patterns of the radioiodinated fatty acids under normal conditions (eg at rest) and during elevated lactate levels (eg during exercise) the differences in distribution must therefore be considered.     

Volatile fatty acids (VFAs) and methane from food waste and cow slurry: Comparison of biogas and VFA fermentation processes

The potential of various biomasses for the production of green chemicals is currently one of the key topics in the field of the circular economy. Volatile fatty acids (VFAs) are intermediates in the methane formation pathway of anaerobic digestion and they can be produced in similar reactors as biogas to increase the productivity of a digestion plant, as VFAs have more varying end uses compared to biogas and methane. In this study, the aim was to assess the biogas and VFA production of food waste (FW) and cow slurry (CS) using the anaerobic biogas plant inoculum treating the corresponding substrates. The biogas and VFA production of both biomasses were studied in identical batch scale laboratory conditions while the process performance was assessed with chemical and microbial analyses. As a result, FW and CS were shown to have different chemical performances and microbial dynamics in both VFA and biogas processes. FW as a substrate showed higher yields in both processes (435 ml CH₄/g VS_fed and 434 mg VFA/g VS_fed) due to its characteristics (pH, organic composition, microbial communities), and thus, the vast volume of CS makes it also a relevant substrate for VFA and biogas production. In this study, VFA profiles were highly dependent on the substrate and inoculum characteristics, while orders Clostridiales and Lactobacillales were connected with high VFA and butyric acid production with FW as a substrate. In conclusion, anaerobic digestion supports the implementation of the waste management hierarchy as it enables the production of renewable green chemicals from both urban and rural waste materials.