Evaluation of Available Energy of Aliphatic Chemicals by Rats

Bioassy procedure to evaluate biologically available energy of chemicals applicable to rats was established, and available energy of 36 chemicals was determined and compared with that estimated by chicks previously. Rats can utilize energy of propionic and butyric acids and n-hexyl propionate and butyrate well, while chicks cannot. Succinic acid, lauryl alcohol and dilauryl succinate at 5% dietary level were available by rats, though at 10% level lauryl alcohol was toxic. Ethyl lactate, octyl and decyl acetates and 1,2-propanediol dilaurate were available by both rats and chicks. Availability of other 6 esters including ethyl succinate and citrate was low. Availability and digestibility of aldehydes by rats were also low.     

Interrelationship between Dilauryl Succinate Feeding and Nutritional Encephalomalacia in Starting Chicks

Various levels of dilauryl succinate with or without additional d-α-tocopheryl acetate and of diethyl succinate were fed to chicks for 4 weeks to examine the interrelationship between the esters and nutritional encephalomalacia.

Chicks fed dilauryl succinate at the level higher than 3% died with lesions in the cerebellum. The lesions were prevented by the supplementation of 25 mg or more of d-α-tocopheryl acetate per kg of diet. Median lethal dietary level for males of meat-type and egg-type chicks at 3 weeks of age was 6.3 and 6.0%, respectively. That for females of meat-type at 3 weeks of age was 9.0%, suggesting that males were more sensitive than females. Diethyl succinate did not induce encephalomalacia.     

Evaluation of Nutritive Value of Glycol Esters as Energy Source for Growing Chicks and Rats

Nutritive value of 4 glycol esters, i.e. ethanediol diacetate, 1,2-propanediol diacetate, 1,3-butanediol diacetate and 1,3-butanediol dioctylate, was estimated biologically by feeding the esters to growing chicks and rats. Energy in the esters taken by both chicks and rats was well utilized, though feed intake of the diets containing the esters at high level tended to decrease. Bitter taste of the esters was suspected to be related to low appetite. The acetates were somewhat volatile and released free acetic acid in the diet during storage. These properties of the acetates makes their use for dietary energy source difficult in practical condition.     

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g/L succinic acid with a yield of 0.53 g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.     

Inhibition of succinic acid production in metabolically engineered Escherichia coli by neutralizing agent,organic acids,and osmolarity

The economical viability of biochemical succinic acid production is a result of many processing parameters including final succinic acid concentration, recovery of succinate, and the volumetric productivity. Maintaining volumetric productivities >2.5 g L^?1 h^?1 is important if production of succinic acid from renewable resources should be competitive. In this work, the effects of organic acids, osmolarity, and neutralizing agent (NH₄OH, KOH, NaOH, K₂CO₃, and Na₂CO₃) on the fermentative succinic acid production by Escherichia coli AFP184 were investigated. The highest concentration of succinic acid, 77 g L^?1, was obtained with Na₂CO₃. In general, irrespective of the base used, succinic acid productivity per viable cell was significantly reduced as the concentration of the produced acid increased. Increased osmolarity resulting from base addition during succinate production only marginally affected the productivity per viable cell. Addition of the osmoprotectant glycine betaine to cultures resulted in an increased aerobic growth rate and anaerobic glucose consumption rate, but decreased succinic acid yield. When using NH₄OH productivity completely ceased at a succinic acid concentration of ～40 g L^?1. Volumetric productivities remained at 2.5 g L^?1 h^?1 for up to 10 h longer when K‐ or Na‐bases where used instead of NH₄OH. The decrease in cellular succinic acid productivity observed during the anaerobic phase was found to be due to increased organic acid concentrations rather than medium osmolarity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Advances in protein–amino acid nutrition of poultry

The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn–soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn–soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary l-cysteine (2.5% or higher) is lethal for young chicks, but a similar level of dl-methionine, l-cystine or N-acetyl-l-cysteine causes no mortality. A supplemental dietary level of 3.0% l-cysteine (7× requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks.     

Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica

Biotechnological production of weak organic acids such as succinic acid is most economically advantageous when carried out at low pH. Among naturally occurring microorganisms, several bacterial strains are known to produce considerable amounts of succinic acid under anaerobic conditions but they are inefficient in performing the low‐pH fermentation due to their physiological properties. We have proposed therefore a new strategy for construction of an aerobic eukaryotic producer on the basis of the yeast Yarrowia lipolytica with a deletion in the gene coding one of succinate dehydrogenase subunits. Firstly, an original in vitro mutagenesis‐based approach was proposed to construct strains with Ts mutations in the Y. lipolytica SDH1 gene. These mutants were used to optimize the composition of the media for selection of transformants with the deletion in the Y. lipolytica SDH2 gene. Surprisingly, the defects of each succinate dehydrogenase subunit prevented the growth on glucose but the mutant strains grew on glycerol and produced succinate in the presence of the buffering agent CaCO₃. Subsequent selection of the strain with deleted SDH2 gene for increased viability allowed us to obtain a strain capable of accumulating succinate at the level of more than 45 g L^?1 in shaking flasks with buffering and more than 17 g L^?1 without buffering. The possible effect of the mutations on the utilization of different substrates and perspectives of constructing an industrial producer is discussed. Biotechnol. Bioeng. 2010;107:673–682. © 2010 Wiley Periodicals, Inc.     

Nutritive Value of Sweet Potato as Carbohydrate Source in Poultry Feed

Chicks were fed on diets containing 23% of protein with various levels of energy from 60 to 75%. Total digestible nutrient is used to show the dietary energy level. The relationship between body weight gain (y) of chicks of 4 weeks fed with standard diets and dietary energy level (x) can be shown as:

By substitution of y of chicks fed on unknown diet, x of the diet can be obtained.

With this principle, it was found that the total digestible nutrients of raw and cooked sweet potato were 47% and 65%, respectively. The former is about 3/4 of the value previously estimated by Morimoto. Results of the determination of metabolizable energy of the test diets endorse the reliability of this conclusion.     

The γ‐aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in Synechocystis sp. PCC 6803

A traditional 2‐oxoglutarate dehydrogenase complex is missing in the cyanobacterial tricarboxylic acid cycle. To determine pathways that convert 2‐oxoglutarate into succinate in the cyanobacterium Synechocystis sp. PCC 6803, a series of mutant strains, Δsll1981, Δslr0370, Δslr1022 and combinations thereof, deficient in 2‐oxoglutarate decarboxylase (Sll1981), succinate semialdehyde dehydrogenase (Slr0370), and/or in γ‐aminobutyrate metabolism (Slr1022) were constructed. Like in Pseudomonas aeruginosa, N‐acetylornithine aminotransferase, encoded by slr1022, was shown to also function as γ‐aminobutyrate aminotransferase, catalysing γ‐aminobutyrate conversion to succinic semialdehyde. As succinic semialdehyde dehydrogenase converts succinic semialdehyde to succinate, an intact γ‐aminobutyrate shunt is present in Synechocystis. The Δsll1981 strain, lacking 2‐oxoglutarate decarboxylase, exhibited a succinate level that was 60% of that in wild type. However, the succinate level in the Δslr1022 and Δslr0370 strains and the Δsll1981/Δslr1022 and Δsll1981/Δslr0370 double mutants was reduced to 20–40% of that in wild type, suggesting that the γ‐aminobutyrate shunt has a larger impact on metabolite flux to succinate than the pathway via 2‐oxoglutarate decarboxylase. ¹³C‐stable isotope analysis indicated that the γ‐aminobutyrate shunt catalysed conversion of glutamate to succinate. Independent of the 2‐oxoglutarate decarboxylase bypass, the γ‐aminobutyrate shunt is a major contributor to flux from 2‐oxoglutarate and glutamate to succinate in Synechocystis sp. PCC 6803.     

Maintaining high anaerobic succinic acid productivity by product removal

During dual-phase fermentations using Escherichia coli engineered for succinic acid production, the productivity and viable cell concentration decrease as the concentration of succinic acid increases. The effects of succinic acid on the fermentation kinetics, yield, and cell viability were investigated by resuspending cells in fresh media after selected fermentation times. The cellular succinic acid productivity could be restored, but cell viability continuously decreased throughout the fermentations by up to 80% and subsequently the volumetric productivity was reduced. Omitting complex nutrients in the resuspension media had no significant effect on cellular succinate productivity and yield, although the viable cell concentration and thus the volumetric productivity was reduced by approximately 20%. By resuspending the cells, the amount of succinate produced during a 100-h fermentation was increased by more than 60%. The results demonstrate that by product removal succinic acid productivity can be maintained at high levels for extended periods of time.     

Fed-batch and continuous fermentation ofSelenomonas ruminantium for natural propionic,acetic and succinic acids

An anaerobic fermentation process was developed for production of natural propionic, acetic and succinic acids froml-lactic acid usingSelenomonas ruminantium. Thel-lactic acid was quickly converted to a racemic mixture and there was no enantiomeric preference for further metabolism. The lactic acid was metabolized to propionic, acetic and succinic acids typically in a molar ratio of about 531. However, the ratio of propionate: succinate started high (as much as 221), before declining to as low as 51 after the first 48 h. Nutrients in corn steep liquor and yeast extract were necessary for optimal production of propionic acid. The corn steep liquor and yeast extract were heat stable at neutral pH, but some nutritional qualities were lost when heated at pH 2.4. In fed-batch fermentation on lactic acid 2.0% propionic acid was produced in 48 h and 2.3% in 68 h. A continuous culture operated at a dilution rate of 0.055 h^–1 and a lactic acid feed concentration of 30 gL^–1 had a propionic acid productivity of 0.59 gL^–1h^–1. The steady state results were: lactic acid 0.6%, propionic acid 1.1%, acetic acid 0.50%, and succinic acid 0.33%.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

Biological conversion of wood hydrolysate to succinic acid by Anaerobiospirillum succiniciproducens

Anaerobiospirillum succiniciproducens grew on a minimal salts medium containing wood hydrolysate (equivalent to 27 g glucose l^–1) and, when supplemented with 10 g corn steep liquor l^–1 as a complex nitrogen source, succinic acid at 24 g l^–1 was obtained (yield = 88% w/w glucose). This may therefore be an economical method to produce succinic acid.     

d-arabinose metabolism byBacteroides fragilis strain 2044

During growth in the presence of 1-14C-d-arabinose,Bacteroides fragilis strain 2044 formed labeled succinic, acetic, and propionic acis. Degradation of the acids by the Schmidt reaction revealed that at least 89% of the succinate radioactivity was found in the methylene carbons and that 75% and 84% of the label in propionate and acetate were found in the noncarboxyl carbons of these molecules. No label was found in acetate, propionate, or succinate during growth of strain 2044 in the presence of 5-3H-d-arabinose. Strain 2044 converted radioactivity from 1- or 2-labeled glycolic acid and glycine to succinate by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons. Label from 1- or 2-labeled glycine and 2 but not 1-labeled glycolic acid was found in acetate. Uniformly labeled 14C-glyoxalate gave rise to labeled acetate, but not succinate.Bacteroides fragilis strain 2044 metabolizesd-arabinose by a mechanism involving a 32 cleavage of the molecule.     

Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions

We examined succinic acid production in Escherichia coli AFP111 using dual-phase fermentations, which comprise an initial aerobic growth phase followed by an anaerobic production phase. AFP111 has mutations in the pfl, ldhA, and ptsG genes, and we additionally transformed this strain with the pyc gene (AFP111/pTrc99A-pyc) to provide metabolic flexibility at the pyruvate node. Aerobic fermentations with these two strains were completed to catalog physiological states during aerobic growth that might influence succinate generation in the anaerobic phase. Activities of six key enzymes were also determined for these aerobic fermentations. From these results, six transition times based on physiological states were selected for studying dual-phase fermentations. The final succinate yield and productivity depend greatly on the physiological state of the cells at the time of transition. Using the best transition time, fermentations achieved a final succinic acid concentration of 99.2 g/l with an overall yield of 110% and productivity of 1.3 g/l h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 325–332 DOI: 10.1038/sj/jim/7000250 Received 01 October 2001/ Accepted in revised form 12 March 2002     

Blue LED and succinic acid enhance the growth of Rhodobacter sphaeroides

Rhodobacter sphaeroides is a purple non-sulfur photosynthetic bacteria that participates in the anoxic cycling of carbon both as the primary producer and as the light-stimulated consumers of the reduced organic compounds. In this study, six different organic acids, i.e. acetate, lactate, oxaloacetate, malate, succinate, and citrate, were selected and used to analyze the relationships between the organic acid source and the cell growth. The C₄ compound exhibited an enhanced cell growth compared to the other organic acids, and the growth rate of R. sphaeroides that was grown with 0.03 M succinic acid was significantly 3.2-fold faster than the C₆ compound of 0.03 M citrate. Additionally, the cell growth of R. sphaeroides was enhanced with increasing light intensity, and the growth rate and the dry cell weight of R. sphaeroides that were grown under the light conditions of 15 W/m² were 2.0- and 1.2-fold higher than R. sphaeroides at 3 W/m². Therefore, the high light intensity probably affected the growth of R. sphaeroides. Moreover, the blue-colored light emitting diode (LED) exhibited a highest growth rate and cell concentration of R. sphaeroides among the various types of LEDs, and the enhanced cell growth phenomenon under the blue LED conditions was dramatically stimulated at low concentrations of succinic acid, which was compensatory for succinic acid. Therefore, a high light intensity and a blue LED as the light source were necessary for the enhanced cell growth for the C₄ organic acid, i.e. succinic acid.     

Effect of diet on fatty acid compositions in Sciaenops ocellatus

The role of dietary polyunsaturated fatty acids (PUFAs) on the fatty acid composition of juvenile red drum Sciaenops ocellatus was investigated. Individuals (n = 435) were fed three natural diets (Gulf menhaden Brevoortia patronus, brown shrimp Farfantapenaeus aztecus and Atlantic brief squid Lolliguncula brevis) that had significantly different proximate composition, energy density and PUFA compositions for 40 days. Diets were characterized as containing: high lipid, high protein, high energy and low PUFA (fish‐based), low lipid, low protein, low energy, moderate PUFA (shrimp‐based), and low lipid, high protein, moderate energy and high PUFA levels (squid‐based), respectively. Specimens were collected at days 0, 5, 10, 20 and 40 to evaluate rate of dietary fatty acid composition in tissues. Two‐source mixing models were used to calculate dietary fatty acid accumulation in consumer tissues. Results indicated that juvenile red drum incorporated an average of 35% dietary PUFAs after 5 days. Although relative biomass and dietary proximate composition had an effect upon the dietary fatty acid contribution, red drum averaged 91% incorporation of the five most prevalent PUFAs [18 : 2 (n ? 6), 20 : 4 (n ? 6), 20 : 5 (n ? 3), 22 : 5 (n ? 3) and 22 : 6 (n ? 3)] across all diets after 40 days. Growth varied as a function of diet and rates were higher for individuals fed the squid diet than those fed shrimp or fish diets primarily due to increased levels of protein and PUFAs [including 22 : 6 (n ? 3); 25·8%] in the diet. Red drum fed squid exhibited the greatest increase in average dietary fatty acid contribution by day 5, a trend that continued for the duration of the experiment. Since PUFA composition in red drum was significantly influenced by diet in as few as 5 days and almost completely incorporated into body tissues after 40 days, results from this study support the premise that fatty acids (especially PUFAs) are promising dietary indicators and may be useful for future studies examining trophic relationships of estuarine and marine fishes.     

Succinic acid production from orange peel and wheat straw by batch fermentations of Fibrobacter succinogenes S85

Succinic acid is a platform molecule that has recently generated considerable interests. Production of succinate from waste orange peel and wheat straw by consolidated bioprocessing that combines cellulose hydrolysis and sugar fermentation, using a cellulolytic bacterium, Fibrobacter succinogenes S85, was studied. Orange peel contains d-limonene, which is a well-known antibacterial agent. Its effects on batch cultures of F. succinogenes S85 were examined. The minimal concentrations of limonene found to inhibit succinate and acetate generation and bacterial growth were 0.01%, 0.1%, and 0.06% (v/v), respectively. Both pre-treated orange peel by steam distillation to remove d-limonene and intact wheat straw were used as feedstocks. Increasing the substrate concentrations of both feedstocks, from 5 to 60 g/L, elevated succinate concentration and productivity but lowered the yield. In addition, pre-treated orange peel generated greater succinate productivities than wheat straw but had similar resultant titres. The greatest succinate titres were 1.9 and 2.0 g/L for pre-treated orange peel and wheat straw, respectively. This work demonstrated that agricultural waste such as wheat straw and orange peel can be biotransformed to succinic acid by a one-step consolidated bioprocessing. Measures to increase fermentation efficiency are also discussed.     

Effect of Dietary Protein and Energy Levels on the Growth Rate,Feed Efficiency and Carcass Composition of Chicks

Feeding day-old White Leghorn male chicks 9 different diets for 4 weeks with the orthogonal composite design, we studied the relationship among dietary protein, energy levels and chick’s responses.

The ellipsoid relationship has been found fitted to describe the relationship between dietary composition and weight gain or feed efficiency of chicks, whereas declined planes are fitted to show the relationship between dietary composition and carcass protein or fat levels of chicks. Dietary protein level has great influence on nitrogen retention, but dietary energy level has little effect.

The linear relationship between total digestible nutrients and metabolizable energy in diets is presented.     

Succinate accumulation in man during exercise

It has been demonstrated in several diving vertebrates that succinate, a component of the Krebs cycle, accumulates in blood during breath-hold dives. The production of succinate is thought to result from amino acid catabolism. Our purpose was to determine whether succinate accumulation occurs in man during muscular activity requiring anaerobic energy contribution. Experiments using an endurance athlete included apneic work on an underwater ergometer and treadmill running to exhaustion. During 1 min breath-hold [(V)\dot]\dot V O₂max, venous succinate increased from 42 [(V)\dot]\dot V O₂max and increased succinate from a similar resting value to 93 M×10^–6. Increases in alanine, lactate, and pyruvate were observed for both types of exercise. The findings confirm that succinate accumulation also occurs in man. It was suggested that amino acid catabolism may provide a source of anaerobic energy production in addition to glycolysis. However, the importance of the proposed energy pathway remains to be quantified.