Biotransformation of ferulic acid to acetovanillone using Rhizopus oryzae

Microbial transformation of ferulic acid to acetovanillone was studied using growing cells of Rhizopus oryzae. Ferulic acid was added to the growing medium (0.5 g L^?1) and incubated for 12 days. The progress of formation of metabolites was monitored by GC and GC-MS after extraction with ethyl acetate. The major metabolite was acetovanillone with minor metabolites formed, such as dihydroferulic acid, coniferyl alcohol and dihydroconiferyl alcohol. Traces of metabolites (≤1–3%), such as vanillin, vanillyl alcohol, vanillic acid and phenyl ethyl alcohol, were also produced. Formation of 4-vinyl guaiacol increased from day 1 (12.4%), reaching a maximum on day 4 (31.7%), and reducing to a minimum on day 12 (3.1%). The formation of acetovanillone increased only from day 2 onward, and reached a maximum (49.2%) on day 12. The optimum concentration of ferulic acid to be added into the medium was found to be only 0.5 g L^?1, as any increase in concentration (0.75 and 1.0 g L^?1) precipitated the precursor, resulting in no further degradation.     

Adattamenti fisiologici alla vita « aerea » nel genere Tillandsia

Abstract

Physiological adaptations to « aerial » life in the genus Tillandsia.—Ten « atmospheric » Tillandsia species, with different degree of adaptation to drought, were investigated to verify the occurrence of CAM and to determine their daily transpiration pattern.

Most of the species were collected during the Botanical Expeditions of the Accademia dei Lincei in Latin America in 1969 and 1971. All species had been maintained in the greenhouse of the Naples Botanical Garden during the winter months and in the open air during the warmer months, previous to experiments. The diurnal cycle of transpiration, the malic acid content and cell sap pH of the leaves were studied under outdoor conditions. Curves of malic acid and pH variations typical of CAM have been obtained for all the examined species. Moreover, the transpiration data indicate that stomata are open at night. The rates of transpiration appear to be extremely low. The highest transpiration rates refer to the species of the relatively moister habitats, the lowest to the more xeric species. Results indicate that CAM and low rates of transpiration are ecological adaptations to extremely dry habitats.     

Neutral Constituents of Volatiles in Cultured Broth of Sporobolomyces odorus

Neutral constituents of volatiles in the ether extract of cultured broth of Sporobolomyces odorus AHU 3246 were analyzed by gas chromatography-mass spectrometry and other methods.

Identified compounds were as follows: Methyl, ethyl, isobutyl, n-butyl, isoamyl, n-amyl, benzyl, and β-phenylethyl alcohol; formaldehyde, acetaldehyde, benzaldehyde, phenyl-acetaldehyde, acetone, and methyl ethyl ketone; ethyl formate, ethyl acetate, and di-n-butyl phthalate; γ-decalactone (4-decanolide) and 4-hydroxy-cis-dodecenoic acid γ-lactone (cis-6-dodecen-4-olide). Di-n-butyl phthalate and parts of methyl, ethyl, and n-butyl alcohol and ethyl acetate were thought to be contaminants. γ-Lactones produced by the yeast were determined by GLC.

Although nine strains of six species of carotenoid pigment accumulating yeasts were cultured under the same conditions, neither flavorful smelling nor γ-lactone production detected in their cultured broths.     

Impact of winemaking practices on arginine and citrulline metabolism during and after malolactic fermentation

AIMS: To study arginine degradation and carcinogenic ethyl carbamate precursor citrulline formation during and after malolactic fermentation (MLF). METHODS AND RESULTS: MLF was induced in white wine with two commercial Oenococcus oeni strains under different winemaking conditions regarding the type of alcoholic fermentation (spontaneous, induced) and the lees management (racked, on lees). Arginine degradation and citrulline formation did not occur during malic acid degradation in any treatment. In five of the six treatments in which arginine degradation took place, it occurred 3 weeks after malic acid depletion and significant amounts of citrulline were formed. Presence of yeast lees in wines led to increased citrulline formation. Conclusions: This study suggests that arginine metabolism is inhibited in oenococci at low pH values (< 3.5) and that in the postalcoholic fermentation phase, citrulline formation from arginine degradation can be avoided if MLF is induced by pure cultures of O. oeni with inhibition of the bacterial biomass after malic acid depletion. Residual yeast lees in the wine have been identified as a significant risk factor for increased citrulline formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions drawn from this study allow reducing the risk of carcinogenic ethyl carbamate formation from citrulline excretion by wine lactic acid bacteria.     

Respiration in Organic Acid-Forming Molds

Cytochrome c, coenzyme Q and lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1, 1, 1, 27) in Rhizopus oryzae were studied in order to investigate the connection between the mechanism of lactate formation and terminal respiration.

Cytochrome c was extracted easily and in good yield by the addition of cetyltrimethyl ammonium bromide to mycelial suspensions. It was purified by calcium phosphate gel and Amberlite IRC-50 resin chromatography.

Coenzyme Q was extracted with ethanol, purified by chromatography on silicic acid, and, following crystallization from a mixture of ethanol and methanol, was identified as coenzyme Q₀.

Lactic dehydrogenase was partially purified and some of its properties were investigated.

Rhizopus oryzae at an early growth stage in shake culture produced almost no lactate. At this stage, the mycelia were rich in cytochrome c and FAD. On the contrary, those of later growth stages fermented a larger amount of the glucose to lactate and the contents of cytochrome c and FAD were lower than in the young mycelia.

Surface cultures produced lactate at a rate very nearly equivalent to the rate of glucose consumption. Addition of zinc to the medium resulted in decreased lactate production, but no increase was observed in the mycelial content of either cytochrome c or FAD in this case. On the other hand, increased quantities of FMN were found in mycelia from shake or surface cultures when zinc was added.     

Ulteriori indagini sul rapporto tra nichel e acidi malico e malonico in Alyssum

Abstract

Further contribution on the relationship between nickel and malic and malonic acids in Alyssum.—Further research has been carried out on extracts of leaves and seeds of A. bertolonii Desv., grown on serpentine and normal garden soil, in order to investigate the nature of the nickel compounds present in this plant. On the purified extracts, obtained with water and formic acid, malic and malonic acids have been determined through gas-chromatography of the trimethyl-sililderivatives; Ni, Ca, Mg and K have been analysed by atomic absorption spectrophotometry.

Both malic and malonic acids are present in the leaves of the plants collected on serpentine and therefore rich in nickel, whilst they are present only in small amounts in the plants grown in garden soil. Seeds contain mainly high levels of malic acid. Remarkable amounts of malic acid, related to a high nickel content, have been observed also in the leaves of another nickel accumulator in the genus Alyssum, i.e. A. serpyllifolium Desf. ssp. lusitanicum Dudley et P. Silva, endemic of Portugal serpentines.     

High levels of malic acid production by the bioconversion of corn straw hydrolyte using an isolated Rhizopus delemar strain

The microbial fermentation of malic acid, which is one of the most important organic acid platforms used widely in food and chemical engineering, has attracted considerable interest. A malate production strain was isolated, a mutation was induced, and regulation of the metabolic network was then conducted. The identification results showed that the malic acid production strain, HF- 119, belonged to Rhizopus delemar. An analysis of the metabolic pathway showed that the malic acid flux of this strain occurred through three main pathways, and many byproducts, such as succinic acid, fumaric acid and ethanol, were produced. Although corn straw hydrolyte was used, the metabolism of xylose was not as rapid as that of glucose. Subsequently, breeding of the strains and regulation of the metabolic network resulted in an increase in malate yield, and the strain HF-121 produced more than 120 g/L malic acid within 60 h. The ability to produce malic acid from biomass hydrolyte highlights the industrial development potential of this strain.     

Linoleic acid, α-linolenic acid,and monolinolenins as antibacterial substances in the heat-processed soybean fermented with Rhizopus oligosporus

ABSTRACT

Antibacterial activities against Staphylococcus aureus and Bacillus subtilis were found in an ethanol fraction of tempe, an Indonesian fermented soybean produced using Rhizopus oligosporus. The ethanol fraction contained free fatty acids, monoglycerides, and fatty acid ethyl esters. Among these substances, linoleic acid and α-linolenic acid exhibited antibacterial activities against S. aureus and B. subtilis, whereas 1-monolinolenin and 2-monolinolenin exhibited antibacterial activity against B. subtilis. The other free fatty acids, 1-monoolein, monolinoleins, ethyl linoleate, and ethyl linolenate did not exhibit bactericidal activities. These results revealed that R. oligosporus produced the long-chain polyunsaturated fatty acids and monolinolenins as antibacterial substances against the Gram-positive bacteria during the fungal growth and fermentation of heat-processed soybean.     

l-Malic Acid Fermentation by Mixed Culture of Rhizopus arrhizus and Proteus vulgaris

When Rhizopus arrhizus NRRL 1526 was mix-cultured with Proteus vulgaris AHU 1144, a strain having a high fumarase activity, in a medium containing glucose as a substrate, fumaric acid fermentation was successively converted to l-malic acid fermentation and large amounts of l-malic acid were accumulated as an end product.

As an inoculum of P. vulgaris for this fermentation, cells in the stationary growth phase (48 to 72 hr culture) were much more favorable than those in the exponential growth phase (18 hr culture) and malic acid yields in the former case were as high as about 70 to 75 % based on initial glucose after 3 to 4 days of the mixed culture.     

Recent advances in microbial production of malic acid from renewable byproducts

In the last few years, ecofriendly malic acid production has received a potential platform for the bio-based chemicals to replace the dependency of fossil based resources. The main goal of this paper is to explore the feasibility of efficient production of malic acid from cost effective alternative renewable byproducts as feedstock. To replace the traditional method of malic acid production from petroleum-based compounds such as maleic acid, the efficiency of fermentation technology for malic acid production using various microorganisms has been improved. To date, glucose is designated as the best substrate for malic acid production. However, few reviews concerning about malic acid production by employing various microbial strains were reported. The current knowledge on the biosynthesis of malic acid has assisted to improve malic acid production using various microbial strains. But, there is still need for the continuous production and replacement of low-cost substrates to increase the yield of malic acid. This review provides an overview about progress, achievements, merits, challenges and future perspectives in malic acid production from cost effective alternative substrates. Thus, malic acid production can be economical using renewable byproducts like crude glycerol by employing appropriate microorganism.

     

Correlation between CAM-Cycling and Photosynthetic Gas Exchange in Five Species of Talinum (Portulacaceae)

Photosynthetic gas exchange and malic acid fluctuations were monitored in 69 well-watered plants from five morphologically similar species of Talinum in an investigation of the ecophysiological significance of the Crassulacean acid metabolism (CAM)-cycling mode of photosynthesis. Unlike CAM, atmospheric CO₂ uptake in CAM-cycling occurs exclusively during the day; at night, the stomata are closed and respiratory CO₂ is recaptured to form malic acid. All species showed similar patterns of day-night gas exchange and overnight malic acid accumulation, confirming the presence of CAM-cycling. Species averages for gas exchange parameters and malic acid fluctuation were significantly different such that the species with the highest daytime gas exchange had the lowest malic acid accumulation and vice versa. Also, daytime CO₂ exchange and transpiration were negatively correlated with overnight malic acid fluctuation for all individuals examined together, as well as within one species. This suggests that malic acid may effect reductions in both atmospheric CO₂ uptake and transpiration during the day. No significant correlation between malic acid fluctuation and water-use efficiency was found, although a nonsignificant trend of increasing water-use efficiency with increasing malic acid fluctuation was observed among species averages. This study provides evidence that CO₂ recycling via malic acid is negatively correlated with daytime transpirational water losses in well-watered plants. Thus, CAM-cycling could be important for survival in the thin, frequently desiccated soils of rock outcrops on which these plants occur.     

Carbon requirements of Colletotrichum gloeosporioides PENZ

Monosporic isolates of Colletotrichum gloeosporioides were made from Punica grantum and one suitable isolate was selected for further work on carbon nutrition. Preliminary experiments showed that a pH value of 5, a temperature of 32°C and a period of 14 days were optimum conditions for the growth of this pathogen. Out of 41 carbon compounds tested, the pathogen showed excellent growth on starch, maltose, melibiose, dextrose, sucrose, raffinose, and dulcitol; good on tartaric acid, mannose, galactose, fructose, mannitol, and castor oil; fair on inulin, isopropyl alcohol, coconut oil, and pectin; poor on sorbose, n-butyl alcohol, arabionose, maleic acid, ethyl alcohol, succinic acid, citric acid, ribose, and malic acid, and no growth on the rest of the carbon compounds. In general, compounds which supported the best mycelial growth, yielded excellent or good sporulation of C. gloeosporioides and vice versa.     

Metabolism of Slices of the Tomato Stem

The rate of respiration of tomato stem slices varied considerably,the highest values (Qo₂, 2–3) being obtained for plantsin a good nutritional state, and the lowest (Qo₂, 1) in starvedplants. The respiratory quotient of 1.0 remained constant. Glucose fermentation was found to follow both glycolytic andalcohol fermentation pathways, the ratio of ethyl alcohol: lacticacid being 6.6:1. Fermentation seems to take place accordingto the Embden-Meyerhof scheme, as shown by the presence of someof these enzymes operative in this scheme and by inhibitionexperiments. In the presence of oxygen there was no formationof alcohol or lactic acid. Pyruvate added to tomato stem slices was metabolized by directoxidation to acetic acid and by dismutation to lactic and aceticacids and CO₂ The metabolism of acetic acid was demonstratedby its condensation with oxaloacetic acid to form citrate, thisbeing the second time that synthesis of citric acid by thismechanism has been found in plants. The presence of aconitase,of isocitric dehydrogenase, of succinic dehydrogenase, and ofmalic dehydrogenase, as well as the inhibition of respirationby malonic acid, favour the hypothesis that oxidation of carbohydratein tomato stem slices proceeds via the citric acid cycle. Thepossibility of an auxiliary route, the malic acid oxidationpathway, also was demonstrated. Tomato stem tissue anaerobicallysplit malic acid into glycolic acid. The further oxidation ofglycolic, glyoxylic, and formic acid was demonstrated. In experiments with C¹⁴-labelled acetate and butyrate a dilutionof the C¹⁴-labelled acids was found after incubation indicatingnew formation of these acids and of active participation offatty acid metabolism in the metabolic activities of the tissue. With the exception of alanine, added amino acids produced adefinite increase in O₂ uptake without extra formation of ammonia. Experimental demonstration of the possibility of electron transportfrom substrate to molecular oxygen in respiration via polyphenoloridase was provided by the attainment in a tomato tissue homogenateof a coupled oxidation-reduction between -ketoglutarate andcatechol with DPN and tyrosinase as the catalysts. The presenceof cytochrome oxidase was also demonstrated. Thus both systemspossibly may take part in the respiration of tomato stem slices.     

Biochemical Aspects of Fumaric Acid Accumulation by Rhizopus arrhizus

The accumulation and excretion of fumaric acid, and to a lesser extent malic and succinic acids, by Rhizopus arrhizus occurs under aerobic conditions in a high-glucose medium containing a limiting amount of nitrogen and a neutralizing agent (CaCO₃). An overall four-carbon dicarboxylic acid molar yield of up to 145% (moles of acid produced per mole of glucose utilized) is obtained after incubation for 4 to 5 days. Evidence is presented that fumarate is synthesized from pyruvate via a carboxylation reaction yielding oxaloacetate, which is then converted to malate and further on to fumarate via the reductive reactions of the tricarboxylic acid cycle. The possible formation of fumarate from the normal (oxidative) operation of the tricarboxylic acid cycle was not excluded by the data. Yield, ¹³C nuclear magnetic resonance, and enzymatic activity studies were carried out in a strain of R. arrhizus which produces high levels of fumarate from glucose and carbonate. The observed high fumarate molar yield (greater than 100%) can therefore be explained in terms of the carboxylation of pyruvate and the operation of the reductive reactions of the tricarboxylic acid cycle under aerobic conditions.     

Metabolic engineering of Rhizopus oryzae: Effects of overexpressing fumR gene on cell growth and fumaric acid biosynthesis from glucose

Fumaric acid is a dicarboxylic acid used extensively in synthetic resins, food acidulants, and other applications, including oil field fluids and esters. The filamentous fungus Rhizopus oryzae is known for its ability to produce and accumulate high levels of fumaric acid under aerobic conditions. In this work, the overexpression of native fumarase encoded by fumR and its effect on fumaric acid production in R. oryzae were investigated. Three plasmids containing the endogenous fumR gene were constructed and used to transform R. oryzae, and all transformants showed significantly increased fumarase activity during both the seed culture (growth) and fermentation (fumaric acid production) stages. However, fumarase overexpression in R. oryzae yielded more malic acid, instead of fumaric acid, in the fermentation because the overexpressed fumarase also catalyzed the hydration of fumaric acid to malic acid. The results suggested that the overexpressed fumarase, encoded by fumR, by itself was not responsible for the over-production of fumaric acid in R. oryzae.     

Genome editing to generate nonfoam-forming sake yeast strains

ABSTRACT

Mutations frequently occur during breeding of sake yeasts and result in unexpected phenotypes. Here, genome editing tools were applied to develop an ideal nonfoam-forming sake yeast strain, K7GE01, which had homozygous awa1?/awa1? deletion alleles that were responsible for nonfoam formation and few off-target mutations. High-dimensional morphological phenotyping revealed no detectable morphological differences between the genome-edited strain and its parent, while the canonical nonfoam-forming strain, K701, showed obvious morphological changes. Small-scale fermentation tests also showed differences between components of sake produced by K7GE01 and K701. The K7GE01 strain produced sake with significant differences in the concentrations of ethyl acetate, malic acid, lactic acid, and acetic acid, while K701 produced sake with more differences. Our results indicated genuine phenotypes of awa1?/awa1? in sake yeast isolates and showed the usefulness of genome editing tools for sake yeast breeding.     

Genetic diversity in <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> strains as revealed by the sequence of lactate dehydrogenase genes

Twenty-seven strains of Rhizopus oryzae accumulating predominantly lactic acid were shown to possess two ldh genes, ldhA and ldhB, encoding NAD-dependent lactate dehydrogenases. Variation in nucleotide sequence was identified for each gene from different strains, and similar phylogenetic trees were obtained based on the nucleotide sequences of both genes. The other 21 strains of R. oryzae accumulating predominantly fumaric and malic acids contained a single ORF of ldhB. Compared to the strains accumulating predominantly lactic acid, a lower degree of sequence divergence was found in ldhB, resulting in a separate cluster in the phylogenetic tree. The high similarity (>90%) spanning the ORF and adjacent regions demonstrates that ldhA and ldhB are derived from the same ancestor gene. The strains accumulating predominantly fumaric and malic acids lack functional ldhA, which plays a role in lactic acid synthesis and may form a lineage separated from the strains accumulating predominantly lactic acid in the genus Rhizopus.     

RELATIONSHIP BETWEEN PLOIDAL LEVEL AND CAM IN FIVE SPECIES OF TALINUM (PORTULACACEAE)

Plants (N = 102) from 23 populations of Talinum, representing five morphologically similar species, were examined in a test of the hypothesis that ploidal level may be correlated with levels of Crassulacean acid metabolism. When grouped by species and ploidal level, two groups of tetraploid plants, T. calcaricum and tetraploid individuals of T. calycinum, had significantly greater malic acid accumulations than any other group, including diploid individuals of T. calycinum. Another tetraploid, T. teretifolium, did not show greater malic acid fluctuations than the diploids, including two putative ancestors (T. mengesii and T. parviflorum). Similarly, malic acid fluctuations in tetraploid individuals of T. parviflorum were not different from diploid individuals of the same species. Nonetheless, when all plants were compared, nocturnal accumulations of malic acid in tetraploid plants were significantly greater than those of diploid plants.     

Esterification Rates of Main Organic Acids in Wines

The esterification rates (ratio of the concentration of an acid in the neutral ethyl ester form to total concentration of the acid) of main organic acids in wines were determined to study the extent of ethyl ester formation of organic acids. The esterification rates ranged from zero to 24.6%. The averaged values of table wines were from 6 to 16% for acetic and lactic acid, from 0.3 to 3.6% for succinic and malic acid, and from zero to 0.1% for tartaric acid. Sherries had higher esterification rates, about 1.6 to 6 times larger, than table wines. It was found that storage time and temperature influence the formation of ethyl esters, and it was suggested that the aging period required for the ester equilibrium is about one year for acetic and lactic acid, and more than two years for succinic, malic and tartaric acid. The possibility and the procedure to control wine quality during the aging process were discussed.     

Photosynthetic Carbon Assimilation in a Shootless Orchid, Chiloschista usneoides (DON) LDL: A Variant on Crassulacean Acid Metabolism

Photosynthetic carbon assimilation in the roots of a shootless orchid Chiloschista usneoides (DON) LDL involves the synthesis and accumulation of malic acid from CO₂ in darkness. Malic acid is consumed in the light.

The roots do not possess stomata or any means of diurnally regulating the diffusive conductance of the pathway between the internal gas phase of the plant and the atmosphere. Regulation of internal CO₂ concentration near to atmospheric levels avoids a large net loss of CO₂ to the atmosphere during malic acid consumption in the light.

The water-absorbing function of the velamen conflicts with the photosynthetic function of the roots. Plants with water-saturated velamina do not acquire CO₂ from the atmosphere at night.