Fermentative Production of Succinic Acid from n-Paraffin by Candida brumptii IFO 0731

An investigation of succinic acid production from n-paraffin under various culture conditions was carried out with Candida brumptii IFO 0731. Ammonium nitrogen was required for both cell growth and succinic acid production. Favorable culture conditions for succinic acid production were ascertained. The productivity was markedly increased by the additions of CaCO₃ and organic nutrients. Under the best condition, the largest quantity of succinic acid production, 23.6 mg/ml, was obtained in a 67% yield from super heavy n-paraffin after 8 days cultivation.     

Transient Accumulation of Fatty Alcohols by n-Paraffin-grown Microorganism

In the course of the study on glutamic acid fermentation by Arthrobacter paraffineus, in which n-paraffin was used as the sole source of carbon, it was observed that fatty alcohollike lipid was accumulated in the paraffin layer of culture medium. This was isolated and identified as the primary fatty alcohol having the corresponding carbon skeleton to that of n-paraffin used as the carbon source.

The accumulation of fatty alcohol occurred rapidly in the early-log phase. The maximum amount of the accumulation was approximately 0.5 mg/ml after 6 hr incubation. In contrast with the production of glutamic acid and trehalose, the addition of penicillin gave no effect on the accumulation of fattv alcohol.

Acetone-treated cells of the n-paraffin-grown bacterium still had the oxidative activity of n-paraffin, and the formation of fatty alcohol from n-paraffin was observed only by the reaction in alkaline pH.     

Trehalose Lipid and α-Branched-β-hydroxy Fatty Acid Formed by Bacteria Grown on n-Alkanes

A bacterium isolated in our laboratory, Arthrobacter paraffineus KY 4303. when grown on n-paraffin as the sole source of carbon, produced anthrone-positive lipid in the emulsion layer (holding bacterial cells, lipids and n-paraffin remained) of the culture medium. This was isolated and identified as α-branched-β-hydroxy fatty acid trehalose ester.

The addition of penicillin to the growing culture caused a significant suppression of trehalose lipid formation and led consequently to the accumulation of both the precursors, α, α-trehalose and α-branched-β-hydroxy fatty acid, in the culture medium.

The formation of trehalose lipid was also observed in other bacteria which can utilize n-paraffin as the sole source of carbon. In addition, a possible role of this trehalose lipid in the utilization of n-paraffin by these bacteria was discussed.     

Production of Antibacterial Compounds Analogous to Chloramphenicol by a n-Paraffin-grown Bacterium

Corynebacterium sp. KY 4339, when grown on n-paraffin (a mixture of C–12 to C–14 fractions) as the sole carbon source, produced three kinds of antibacterial compounds which were tentatively named Corynecins. These compounds were isolated by the extraction from the culture broth with ethyl acetate and by the chromatographies on silicic acid and alumina columns. Each component demonstrated some similarity to chloramphenicol on thin-layer chromatogram. Although their biological activities were not so remarkably as that of chloramphenicol, the patterns of antibacterial spectra against gram-positive and gram-negative bacteria resembled to it.

For the production of corynecins, n-paraffin was a preferable carbon source. By controlling the pH of the medium in the neutral range and keeping the aeration at a high level during the fermentation, approximately 3 g of corynecins per liter of the medium were produced after 72-hr incubation.     

Studies on the Utilization of Hydrocarbons by Microorganisms

Fumaric acid productivity of Candida hydrocarbofumarica in various culture conditions was investigated. Namely, the effects of pH, heavy metal ions, hydrocarbon concentration, aeration and surface active agents were studied.

The addition of CaCO₃ and the aeration were effective for fumaric acid production.

The rate of conversion of n-paraffin to fumaric acid gradually decreased as the concentration of n-paraffins (6%) was increased.

A very high yield, 84% was obtained with a culture medium containing 6% of n-paraffins for 7 days culture.     

Metabolic engineering of Mannheimia succiniciproducens for succinic acid production based on elementary mode analysis with clustering

Mannheimia succiniciproducens, a capnophilic gram‐negative rumen bacterium, has been employed for the efficient production of succinic acid. Although M. succiniciproducens metabolism was previously studied using a genome‐scale metabolic model, more metabolic characteristics are to be understood. To this end, elementary mode analysis accompanied with clustering (‘EMC’ analysis) is used to gain further insights on metabolic characteristics of M. succiniciproducens allowing efficient succinic acid production. Elementary modes (EMs) generated from the central carbon metabolic network of M. succiniciproducens are clustered to systematically analyze succinic acid production routes. Based on the results of EMC analysis, zwf gene is identified as a novel overexpression target for the improved succinic acid production. This gene is overexpressed in a previously constructed succinic acid‐overproducing M. succiniciproducens LPK7 strain. Heterologous NADPH‐dependent mdh is later intuitively selected for overexpression to synergistically improve succinic acid production by utilizing abundant NADPH pool mediated by the overexpressed zwf. The LPK7 strains co‐expressing mdh alone and both zwf and mdh genes are subjected to fed‐batch fermentation to better examine their succinic acid production performances. Strategies of EMC analysis will be useful for further metabolic engineering of M. succiniciproducens and other microorganisms to improve production of succinic acid and other chemicals of interest.     

Extracellular Accumulation of Organic Acids by Isopropanol-utilizing Microorganisms

Isopropanol-utilizing microorganisms were newly isolated from soils and several of them accumulated two acids in the culture broth, α-ketoglutaric acid being a major one and succinic acid a minor one. Two strains (N–79 and S–1), classified as the genus Mycobacterium, were examined for the cultural conditions with respect to the accumulation of the acids. The accumulation of α-ketoglutaric acid depended greatly on the pH value in the broth, which is required to be kept at around 4 for the maximum accumulation. By means of the pH-controlled culture (at 3.5) with a jar fermentor, strain N–79 accumulated α-ketoglutaric acid at a rate of 0.015 g/liter/hr. The data obtained in this work indicate that the metabolism of isopropanol by strain N–79 probably proceeds via the acetone pathway without the inter-conversion between isopropanol and n-propanol.     

Systems approaches to succinic acid-producing microorganisms

Succinic acid is a cellular metabolite belonging to the C4-dicarboxylic acid family, and the fermentative production of succinic acid via the use of recombinant microorganisms has recently become the focus of an increasing amount of attention. Considering the difficulty inherent to the direct application of natural succinic acid producers to the industrial process, a variety of systems biology studies have been conducted regarding the development of enhanced succinic acid production systems. This review shows how the metabolic processes of microorganisms, includingEscherichia coli andMannheimia succiniciproducens, have been optimized in order to achieve enhanced succinic acid production. First, their metabolic networks were constructed on the basis of complete genome sequences, after which their metabolic characteristics were estimated viain silico computer modeling. Metabolic engineering strategies were designed in accordance with the results ofin silico modeling and metabolically engineered versions of bothE. coli andM. succiniciproducens have been constructed. The succinic acid productivity and yield obtained using metabolically engineered bacteria was significantly higher than that obtained using wild-type bacteria.     

Selective extraction of succinic acid from binary mixture of succinic acid and acetic acid

In production of succinic acid by fermentation, succinic acid and acetic acid are co-produced. To purify the succinic acid from binary-acid mixture of succinic acid and acetic acid, the tertiary amine-based extraction was used. In 1-octanol, the selectivity for succinic acid was proportional to the chain length of tertiary amine. But, the distribution of acids into organic phase was low in n-heptane. These results are due to the different degree of intramolecular hydrogen bonding of succinic acid and hydrophobicity of each acid.     

Carboxylic Acids in Cultured Broth of Sporobolomyces odorus

Carboxylic acids found in the cultured broth of Sporobolomyces odorus AHU 3246 which produces γ-lactones as principles of the aromatic flavor, were analyzed. The concentrate of methylated acids was steam-distilled and in the residue, succinic acid, nonanedioic acid (azelaic acid), undecanedioic acid and 2-hydroxy-3-phenylpropionic acid (β-phenyllactic acid) were identified as their methyl esters by GLC and spectroscopic methods. Phthalic acid and its mono-n-butyl ester were also found, but these compounds were thought to arise from di-n-butyl phthalate, one of impurities of deionized water.     

Studies on the Lipids of Scale Insects

Corynebacterium cultures, isolated from soil by the use of n-paraffin as the sole carbon source have been shown to bring about significant oxidation of a new compound, dl-cistetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI), in the presence of hydrocarbon.

The co-oxidation products were isolated from culture broth and identified as dl-biotinol and dl-biotin by IR, NMR and MS spectroscopies.     

Poduction of Biotin by Microbial Transformation of dl-cis-Tetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI)

The conditions for biotin production were investigated. Urea was a more effective nitrogen source than ammonium chloride and ammonium sulfate. About 60% conversion from dl-cis-tetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI) to biotinol and biotin occurred using Corynebacterium sp. B–321. Strain M–6318 which derived from B–321 as a mutant incapable of assimilating n-alkane produced large amounts of dl-biotin from dl-TOPTI. The inability of the microbe to assimilate n-alkane resulted in repression of biotin degradation. Maximum conversion (80%) was obtained by growing cultures of strain M–6318 in the constant presence of n-paraffin.     

L-Threonine Production by a Mutant of Arthrobactev paraffineus

An isoleucine leaky auxotroph of Arthrobacter paraffineus, which was isolated by Takayama et al.³⁾ as a mutant producing L-threonine and L-valine from n-paraffin, was subjected to further mutagenesis in an attempt to obtain better L-threonine producers. Some of the double auxotrophs derived from the isoleucine auxotroph and some of their revertants with respect to isoleucine requirement produced more L-threonine than the original isoleucine auxotroph. In contrast to the original isoleucine auxotroph, a revertant derived from a methionine plus isoleucine double auxotroph, KY7135, produced an increased amount of L-threonine and a decreased amount of L-valine. The optimum level of L-methionine for L-threonine production in KY7135 was much higher (1000 ~ 2000 μg/ml) with n-paraffin medium than with sorbitol or mannitol medium (10 ~ 50 μg/ml). L-Threonine production reached a maximum level (11.5 mg/ml) in 7 days incubation with the medium containing 10% n-paraffin (C₁₂ ~ C₁₄ rich). Several mutants which produce L-threonine more than 12 mg/ml were obtained from KY 7135 by monocolony isolation procedure.     

Emulsification and degradation of "Bunker C" fuel oil by microorganisms

An enrichment culture procedure has been used to isolate mixed culture systems which grow upon “Bunker C” fuel oil. When inoculated into a mineral salts aqueous medium containing Bunker C oil, the mixed cultures initiate oil emulsification. Emulsification usually is observed in 24–48 hr. The role of microbes in this emulsification will be discussed. It appears that certain metabolic products produced by the microbe possess properties of surfactants. Bacteria and fungi have been isolated which possess the ability to cause emulsification. Freeze-dried biomass is also capable of emulsifying oil. Chromatographic analyses of biodegraded Bunker C fuel oil show that microorganisms selectively metabolize the n-paraffin fraction.     

The Structures of Corynecins; Chloramphenicol Analogues Produced by a n-Paraffin-grown Bacterium

Corynebacterium sp. produced antibacterial substances (corynecins) when a n-paraffin mixture was used as the sole carbon sourse. Structures of the substances were determined as acyl derivatives of d-(?)-threo-l-p-nitrophenyl-2-amino-l,3-propanediol (free base of chloramphenicol).     

Availability of Energy in Esters of Aliphatic Acids and Alcohols by Growing Chicks

Biological availability of 106 esters of alcohols and aliphatic mono-, di- and tri-carboxylic acids and diethylene glycol succinate was compared by the mini-test with chicks. Chicks can utilize methyl esters of saturated fatty acids of carbon chain from 10 to 14, ethyl esters of those from 9 to 12, propyl caprate, n-butyl esters of those from 8 to 12, n-amyl esters of those from 6 to 12, n-hexyl n-butyrate and i-vaterate, and n-octyl and n-decyl acetates. Only 3 dicarboxylates, i.e. di-octyl and di-lauryl succinates and di-methyl cis-cyclopropane-l,2-dicarboxylate, were available among the dicarboxylates tested. Availability of ethyl esters of succinic, fumaric and citric, acid was unexpectedly low.     

Utilization of gas oil by a yeast culture

A mixed yeast culture (Culture 4) was grown on representative gas oil samples as well as paraffin wax. Culture 4 was found to utilize n-paraffinic hydrocarbons almost quantitatively from most gas oil fractions; significant alteration of other hydrocarbon components was not detected. Generation times of 4.0–9.0hr. were typical during the exponential growth phase in fermentations with various gas oil fractions. Cell yields were 70–90% based on n-paraffin utilization. The culture appeared to exhibit maximum efficiency of n-alkane removal in the C₁₉ to C₂₄ range. The cells recovered from the fermentations contained 8.8–9.3% nitrogen. Paraffin wax also served as a suitable carbon source when dissolved in 2,6,10,14-tertramethylpentadecane (pristane). However, substrate utilization appeared to be incomplete.     

n-Dodecane as a substrate for nitrogen fixation by an alkane-utilizingAzospirillum sp.

Azospirillum sp. ANK-BI-11 was isolated from petroliferous soil. Glucose, nutrient broth, and sugar acids showed better growth thann-alkanes under aerobic conditions. The utilization of glucose was inhibited in the presence ofn-hexane. Microaerobically, succinic acid, pyruvic acid, and lactic acid were the best C-sources for acetylene reduction, whereas glucose was the best source for growth.n-Dodecane, a nonconventional C-source, also showed good response towards acetylene reduction, although growth was not so pronounced here as with glucose but was equal to that of Na-succinate. Optimum pH and temperature for acetylene reduction were between 7.0 and 8.0 and 30°C, respectively. Scanning electron microscopic studies revealed structural alteration in the shapes and sizes of the cells ofAzospirillum sp. when grown onn-hexane andn-dodecane compared with the cells grown on glucose.     

Fermentation of n-Paraffins by Yeast

Screening test for obtaining microorganisms which produce l-amino acids or organic acids from n-paraffins were carried out. Fourteen strains of microorganisms which seemed to belong to the yeast showed strong ability to produce α-ketoglutaric acid. A representative strain of these microorganisms was identified as Candida lipolytica AJ 5004.

Optimal conditions for production of α-ketoglutarate using Candida lipolytica AJ 5004 were also studied. Under the condition thus obtained using a culture medium of 8 weight % of n-paraffins, the yeast accumulated 59% of α-ketoglutarate to the substrate added after three days culture.     

Extracellular Accumulation of DNA by Hydrocarbon-utilizing Bacteria

Some hydrocarbon-utilizing bacteria grown on n-paraffin as the sole source of carbon accumulated extracellularly a considerable amount of DNA (0.1 g to 0.6 g per liter) which was free from intact cells and slime materials. This was particularly noted when the strains belonging to Pseudomonad were employed. n-Paraffin was a preferable carbon source for the accumulation of DNA by Pseudomonad, while a strain of Arthrobacter accumulated DNA by growing it on glucose.

The DNA thus accumulated was easily isolated and purified free from other cellular components. The purified DNA was highly polymerized (above 4 × l0⁶ daltons), having the same base composition as the cellular DNA.