Microcalorimetric study of Escherichia coli aerobic growth: theoretical aspects of growth on succinic acid.

Two methods of investigation were used to evaluate the heat quantity associated with anabolic processes (qan) during the aerobic growth of Escherichia coli in a minimal medium containing succinic acid as the sole energy and carbon source. The study of the contribution of biosynthetic reactions from succinic acid and ammonia were investigated by both methods. The two qan values obtained were in excellent agreement and were found to be significant. Thus it was demonstrated that the contribution of anabolism strongly influenced the quantity of heat associated with microbial aerobic growth. The qan calculated as above explained the experimental enthalpy change which was recently reported.     

Effects of dissolved CO2 levels on the growth of Mannheimia succiniciproducens and succinic acid production

A capnophilic rumen bacterium Mannheimia succiniciproducens produces succinic acid as a major fermentation end product under CO(2)-rich anaerobic condition. Since succinic acid is produced by carboxylation of C3 compounds during the fermentation, intracellular CO(2) availability is important for efficient succinic acid formation. Here, we investigated the metabolic responses of M. succiniciproducens to the different dissolved CO(2) concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO(2) concentration was below 8.74 mM. On the other hand, cell growth and succinic acid production increased proportionally as the dissolved CO(2) concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO(2) concentration of 141 mM were 1.49 and 1.52 times higher, respectively, than those obtained at the dissolved CO(2) concentration of 8.74 mM. It was also found that the additional CO(2) source provided in the form of NaHCO(3), MgCO(3), or CaCO(3) had positive effects on cell growth and succinic acid production. However, growth inhibition was observed when excessive bicarbonate salts were added. By the comparison of the activities of key enzymes, it was found that PEP carboxylation by PEP carboxykinase (PckA) is the most important for succinic acid production as well as the growth of M. succiniciproducens by providing additional ATP.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

Amino acid limited growth of starter cultures in milk

The specific growth rates of several Streptococcus cremoris strains were 10–40% lower in milk than in other growth in media. The growth rates in milk increased when an amino acid mixture or casein was added, whereas, when milk was diluted, the specific growth rate of the streptococci decreased. This decrease could be overcome by bringing the casein concentration in the diluted milk back to the normal value (3%). This indicates that casein-hydrolysis proceeded at a rate too low for the streptococci to reach their potential maximum specific growth rates in milk so that growth in milk is essentially amino acid-limited. This was subsequently demonstrated for S. cremoris by continuous cultivation in media with low casein concentrations. At a low dilution rate casein hydrolysis was fast enough to supply the cells with enough amino acids and lactose was growth-limiting, whereas at higher dilution rates amino acids became growth-limiting. In cultures exponentially growing in milk the concentration of free amino acids was measured to determine which amino acid(s) was(were) absent and could possibly limit growth. A number of essential amino acids (leucine, methionine, glutamate and in some cases phenylalanine) were not detected and addition of these, together, stimulated the growth of S. cremoris in milk. The amino acids leucine and phenylalanine appeared to play a particularly important role in this stimulation. These two are, supposedly, the first amino acids that become limiting during growth in milk. The effect of competition for casein and amino acids by different organisms was studied in continuous cultures. At different dilution rates different strains became dominant in these mixed cultures, suggesting that differences in apparent affinity constants (K_S) for casein, leucine and glutamate existed between the strains.     

The effect of protectants and pH changes on the cellular growth and succinic acid yield of Mannheimia succiniciproducens LPK7

The harmful effects of succinic acid and oxidative stress on cell growth were determined during batch fermentation with Mannheimia succiniciproducens LPK7, a powerful succinic acid-producing strain, and conditions were optimized to minimize these effects. In terms of toxicity, the cell concentration decreased as the concentration of succinic acid increased. By changing the pH from 6.5 to 7 during fermentation, the cell concentration increased by about 10%, and the level of succinic acid production was 6% higher than that of the control. In addition, by introducing protectants, the cell concentration increased by about 10%, and the level of succinic acid produced was increased by 3%.     

Role of cell-wall biogenesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a growth-limiting protein induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.Abbreviations CHI cycloheximide - COR cordycepin - DCB 2,6-dichlorobenzonitrile - GLP growth-limiting protein(s) - IAA indole-3-acetic acid     

Comparison of Growth and Primary Shunt Product Formation by Claviceps purpurea Cultured on Succinic Acid and Glucose as Carbon Sources

Growth on a medium containing succinic acid as the sole carbon source produced 1 g (dry weight) of mycelium per liter of medium by 50 days of incubation, whereas 25 g of mycelium was produced in 10 days when glucose was also present in the medium. Primary shunt metabolism took place during growth on succinic acid in spite of the extremely slow growth. Mycelia grown on succinic acid contained a higher percentage of residual mycelium and phosphate, but a lower percentage of mannitol, carbohydrate, lipid, and water-soluble nitrogen, than mycelia grown on a mixture of glucose and succinic acid. Thus, although primary shunt metabolism is favored by rapid growth on a rich, balanced sugar medium, it can also take place during extremely restricted growth in a medium containing succinic acid as the sole carbon source.     

Quantitative gas chromatography of Bacteroides species under different growth conditions

From 56 strains of strictly anaerobic gram-negative rods isolated from stool and purulent lesions the fermentation products in the presence and absence of hemin were determined by quantitative gas-solid chromatography, using a simple and more rapid chromatographic procedure. With hemin the fermentation products were propionic, acetic, lactic and succinic acid. Without hemin no or little succinic acid was formed and the main products were lactic and acetic acid. In both groups the distribution of subspecies was determined and the production of fatty acids measured quantitatively.Fourteen strains of the lesion group showed a higher metabolic activity, resulting in an increased total acid production caused by an excessive production of acetic and lactic acid. This characteristic is probably a virulence factor in these strains.All strains were protoporphyrin- and oxgall-dependent. It is postulated that these substances are used for the production of cytochromes which permits the formation of succinic acid by a fumarate reductase resulting in an increased growth rate and growth yield.     

Acid Tolerance of Leuconostoc mesenteroides and Lactobacillus plantarum

In this study, we determined the internal cellular pH response of Leuconostoc mesenteroides and Lactobacillus plantarum to the external pH created by the microorganisms themselves or by lactic or acetic acids and their salts added to the growth medium. Growth of Leuconostoc mesenteroides stopped when its internal pH reached 5.4 to 5.7, and growth of L. plantarum stopped when its internal pH reached 4.6 to 4.8. Variation in growth medium composition or pH did not alter the growth-limiting internal pH reached by these microorganisms. L. plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate or sodium lactate down to an external pH of 3.0 with either acid. In contrast, the DeltapH of Leuconostoc mesenteroides was zero at pH 4.0 with acetate and 5.0 with lactate. No differences were found between d-(-)- and l-(+)-lactic acid for the limiting internal pH for growth of either microorganism. The comparatively low growth-limiting internal pH and ability to maintain a pH gradient at high organic acid concentration may contribute to the ability of L. plantarum to terminate vegetable fermentations.     

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.     

Differential growth rates of Candida utilis mother and daughter cells under phased cultivation.

The yeast Candida utilis was continuously synchronized by the phased method of cultivation with the nitrogen source as the growth-limiting nutrient. The doubling time (phasing period) of cells was 6 h. Both cell number and deoxyribonucleic acid synthesis showed a characteristic stepwise increase during the phased growth. The time of bud emergence coincided with the time of initiation of deoxyribonucleic acid synthesis. Size distribution studies combined with microscopic analysis showed that the cells expanded only during the unbudded phase of growth. Usually the cells stopped increasing in size about 30 min before bud emergence, and the arrest of the increase in cell volume coincided with the exhaustion of nitron from the medium. There was no net change in the volume of cells during the bud expansion phase of growth, suggesting that as the bud expanded, the volume of the mother portion of the cell decreased. After division the cells expanded slightly. The postdivision expansion of cells, unlike the growth before bud initiation, occurred in the absence of the growth-limiting nutrient. The newly formed daughter cells were smaller than the mother cells and expanded at a faster rate, so that both types of cells reached maximum size at the same time. Possible reasons for the different rates of expansion of mother and daughter cells are discussed.     

Studies on aspergilli XVII. Effect of nitrogen sources on growth and fruiting

Summary The effect of ammonium, nitrate, and organic nitrogen on growth and sporulation of 18 Aspergilli was examined in a chemically defined medium in surface culture under controlled conditions. All three forms of nitrogen were metabolized by all the Aspergilli tested. Ammonium nitrogen was not good both for growth and fruiting. This was due to the sharp fall in the pH level which resulted due to the rapid utilization of anions of the ammonium nitrogen than cations. The effect of adding succinic acid in the medium containing ammonium nitrogen has been discussed.Good growth of Aspergilli in media containing nitrate nitrogen with the accompanying rise in the pH of the medium showed that these species are capable of reducing nitrate nitrogen to the level of ammonia. The role of succinic acid in the utilization of nitrate nitrogen was investigated. All fungi accomplished good growth on a medium containing asparagine.     

糠醛和5-羟甲基糠醛对大肠杆菌产丁二酸的影响

利用可再生生物质特别是木质纤维素水解液来生产平台化合物丁二酸,是目前研究的热点。虽然许多研究者相继报道了木质纤维素水解液对菌株生长和丁二酸生产存在一定抑制作用,但并没有水解液中各种抑制物对菌株影响的相关动力学研究及机理研究。我们选择了两种代表性木质纤维素水解液抑制物,即糠醛和5-羟甲基糠醛,系统研究了它们对大肠杆菌的生长和丁二酸生产的影响。结果表明：糠醛和5-羟甲基糠醛的初始抑制浓度均为0.8 g/L。当糠醛浓度大于6.4 g/L,5-羟甲基糠醛浓度大于12.8 g/L时,菌株生长完全受到抑制。在最高耐受浓度下,糠醛的存在使菌株生物量比对照菌株下降77.8%,丁二酸产量下降36.1%。5-羟甲基糠醛的存在使菌株生物量比对照菌株降低13.6%,丁二酸产量降低18.3%。糠醛和5-羟甲基糠醛具有明显的协同作用。体外酶活测定表明丁二酸生产途径中关键酶磷酸烯醇式丙酮酸羧化酶、苹果酸脱氢酶、富马酸还原酶均受糠醛和5-羟甲基糠醛抑制。研究结果对丁二酸生产用纤维素水解液的预处理和脱毒工艺开发具有指导作用,有利于实现丁二酸发酵生产的工业化。     

一株耐受低pH、高浓度乳酸及琥珀酸菌株的筛选与鉴定

【目的】通过构建的人工耐酸系统,筛选耐受低pH值、乳酸及琥珀酸的菌株。【方法】构建人工耐酸系统长期驯化菌株,利用不同p H的酸性平板进行筛选,从环境中筛选出一株对低p H值、高浓度乳酸以及琥珀酸有很好耐受性的菌株。通过形态学特征、生理生化特征研究,并结合18S rDNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类。【结果】经过酸性人工系统的长期驯化,筛选分离出一株耐受低pH值、高浓度乳酸以及琥珀酸的菌株WJ-2,经鉴定该菌株为酿酒酵母(Saccharomyces cerevisiae),其最适生长温度为30°C。酸性平板实验显示该菌株能够耐受pH2.5的酸性环境,同时对9%的乳酸及8%的琥珀酸也有很好的耐受性。另外,耐酸菌株WJ-2在pH 2.5、9%乳酸和8%琥珀酸的培养环境中仍能保持相对中性的细胞内pH值。【结论】通过构建人工酸性系统,成功筛选出一株对低pH值、高浓度乳酸以及琥珀酸具有耐受性的菌株——酿酒酵母菌WJ-2,该方法可为筛选具有特定耐受能力菌株提供一个新思路。     

Effect of yeast extract and succinic acid on growth and sporulation of alternaria species

Summary Alternaria solani andA. nyctanthi, these pathogens causing leafspot disease were able to metabolize a variety of nitrogen compounds when grown on different culture media. The amount of growth varied with the nitrogen source. Peptone produced the best zonation when added in definite proportion to the yeast extract medium. Ammonium compounds were found to be moderately effective for growth but poor for sporulation. The effect of adding succinic acid in media containing ammonium sources and the role of pH in the utilization of nitrite nitrogen was investigated.The fungus gave more vegetative growth on a mixture of aminoacids than in culture media in which the same amino acids were supplied singly to study the effect produced on growth and sporulation.     

Studies on the biochemistry of Penicillium charlesii. Influence of various dicarboxylic acids on galactocarolose synthesis

1. It has been shown that Penicillium charlesii continues to synthesize galactocarolose when l-malic acid, malonic acid, succinic acid, fumaric acid, maleic acid or oxaloglycollic acid is substituted for dl-tartaric acid in the Raulin-Thom nutrient medium. 2. The quantity of galactocarolose synthesized per g. of mycelia was markedly decreased by substitution of l-malic acid, malonic acid, succinic acid, fumaric acid or maleic acid for dl-tartaric acid. Substitution of oxaloglycollic acid for dl-tartaric acid did not depress the galactocarolose synthesized/g. of mycelia; however, the quantity of fungal mass formed was decreased approximately fivefold. 3. Based upon (14)C incorporation into galactocarolose, succinic acid, fumaric acid or malonic acid did not serve as direct precursors of galactose as did tartaric acid. Oxaloglycollic acid, l-malic acid and maleic acid were not tested. 4. The relative quantity of galactocarolose synthesized per g. of mycelia decreased as the concentration of diammonium dicarboxylate added to the growth medium was increased. Tartaric acid, oxaloglycollic acid, fumaric acid and malonic acid were tested. 5. The quantity of mycelia formed and the quantity of galactocarolose synthesized per g. of mycelia were greater when the growth medium contained l-tartrate than when it contained d-tartrate.     

Effects of growth rate and influent substrate concentration on effluent quality from chemostats containing bacteria in pure and mixed culture

Studies were performed using pure cultures of A. acrogenes and E. coli and a heterogeneous microbial population growing in carbon-limited chemostats with glucose as the sole carbon and energy source. A two-level factorial experimental design was employed to test the hypothesis that the concentration of growth-limiting substrate in a chemostat is controlled by the growth rate alone and is independent of the concentration of substrate entering the reactor. The pure culture experiments showed that the conclusions depend upon the measurement employed for growth-limiting substrate. When the concentration of glucose was measured directly, the hypothesis was found to be true within the limits of the study (500–1500 mg/liter). However, if the chemical oxygen demand (COD) test was used as the measure of growth-limiting substrate the hypothesis was found to be false. When heterogeneous cultures were employed the hypothesis was false regardless of the technique used to measure the concentration of growth-limiting substrate. Nevertheless, it was possible to generate regression equations which described the interactions among influent COD, growth rate, and effluent COD with a high level of correlation.     

Effect of growth substrate on thermal death of thermophilic bacteria

The heat sensitivity of gram-negative, hydrocarbon-utilizing thermophilic bacteria was altered by a change in growth substrate. Thermophilic strains CC-6, BI-1, and LEH-1, grown with acetate or n-heptadecane as the carbon source, had a higher survival rate when incubated 5 degrees C above their maximum growth temperature than cells of the same organism after growth on glucose or glycerol. There was a correlation between the growth substrated, heat resistance, and the ratios of cellular n-hexadecanoic acid/branched hexadecanoic acid and n-heptadecanoic acid/branched heptadecanoic acid. The bacterial cells that were more heat resistant had ratios of straight-chain/branched-chain fatty acids above 1.0, whereas the heat-sensitive cells had ratios below 0.6.     

Time course of auxin stimulations of growth

Measurements of the time course of growth responses of corn coleoptile sections to pulses of auxin (10⁻⁵m indoleacetic acid) establish that the growth rate changes in a regular pattern around the auxin pulse: a latent phase of 12 to 15 minutes is followed by an acceleration of growth rate lasting 15 to 20 minutes, after which a fairly steady rate is maintained. When the auxin source is withdrawn, there is an after-effect of about 15 minutes followed by a decay of growth rate, which reaches 50% decay after a further 15 to 40 minutes. The decay phase appears to be a function of the transport of auxin out of the sections. The 50% decay of growth for single cells is estimated at 30 minutes from the time of withdrawal of an exogenous supply of auxin. The regulation of growth by auxin is rapidly imposed or dissipated as auxin enters and exits, respectively, suggesting a facile association and disassociation of auxin with a growth-limiting site in the cell. It is proposed that the growth-stimulated state is dissipated at once when the transportable auxin has passed out of the cell.     

Kinetic analysis of bifidobacterial metabolism reveals a minor role for succinic acid in the regeneration of NAD+ through its growth-associated production

Several strains belonging to the genus Bifidobacterium were tested to determine their abilities to produce succinic acid. Bifidobacterium longum strain BB536 and Bifidobacterium animalis subsp. lactis strain Bb 12 were kinetically analyzed in detail using in vitro fermentations to obtain more insight into the metabolism and production of succinic acid by bifidobacteria. Changes in end product formation in strains of Bifidobacterium could be related to the specific rate of sugar consumption. When the specific sugar consumption rate increased, relatively more lactic acid and less acetic acid, formic acid, and ethanol were produced, and vice versa. All Bifidobacterium strains tested produced small amounts of succinic acid; the concentrations were not more than a few millimolar. Succinic acid production was found to be associated with growth and stopped when the energy source was depleted. The production of succinic acid contributed to regeneration of a small part of the NAD+, in addition to the regeneration through the production of lactic acid and ethanol.