Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes

Microorganisms which produced n-alkane ω,ω′-dicarboxylic acid (DC) from n-alkane were selected from natural sources. It was found that the best three producers thus obtained belonged to yeast. All of the stock cultures which are able to assimilate n-alkane and are belonged to genus Candida and Pichia were also found to produce DC from n-alkane.

Candida cloacae 310, a representative strain selected from natural source, was able to produce DCs having 5 to 16 carbon atoms from various n-alkanes. Among them, DCs with 5 to 9 carbon atoms were more heavily accumulated than those with more than 9, except those with the same number of carbon atoms as the substrates which were the main products from the substrates with less than 15 carbon atoms. It was also clearly demonstrated that DCs with odd carbons alone were produced from n-alkanes with odd carbons, while DCs with even carbons alone from n-alkanes with even carbons.

Then, cultural conditions of Candida cloacae 310 were studied for the production of DC-12 from n-dodecane (n-C₁₂) which showed the highest yield among the observed accumulation.

Under the optimum conditions, 2.28 g/liter of DC-12 was obtained together with 1.86 g/liter of DC-6 and 0.82 g/liter of DC-8 after 72 hr’ cultivation in a synthetic medium containing 100 ml of n-C₁₂ per liter.     

Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes

Strain M-l which was derived from Candida cloacae 310 as a mutant unable to assimilate dicarboxylic acid (DC) produced large amount of DCs from n-alkanes, as expected. It produced DCs with the same number of carbon atoms as those of n-alkanes used (9 to 18 carbon atoms). Among DCs produced, n-tetradecane ω,φ′-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) was most abundantly accumulated and the highest level of DC-16, i.e., 29.3g/liter was obtained by resting cells.

On the other hand, since the growth rate of strain M-l on n-alkane markedly decreased in comparison with that of the parent strain, other carbon source which supported the growth of strain M-l was necessary for the production of DC from n-alkane by growing cells. When acetic acid was used as carbon source for the growth in DC-16 production from n-C₁₆, the highest level of DC-16, i.e., 21.8 g/liter was obtained ofter 3 days' cultivation.     

Production of l-Phenylalanine from trans-Cinnamic Acid with Rhodotorula glutinis Containing l-Phenylalanine Ammonia-Lyase Activity

An enzymatic method using l-phenylalanine ammonia-lyase (EC 4.3.1.5) for the rapid conversion of trans-cinnamic acid to l-phenylalanine has been investigated. With Rhodotorula glutinis, enzyme activity as high as 0.3 U/ml of culture broth was obtained. The enzyme activity was kept stable for a relatively long time during cultivation by the addition of l-isoleucine. Optimization of the parameters of the conversion reaction resulted in accumulation of 18 mg of l-phenylalanine per ml of reaction mixture. The conversion yield from trans-cinnamic acid was about 70%. The method may provide a rapid and practical way to produce l-phenylalanine useful as an essential amino acid.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Methylketones from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Cell suspensions of methane-utilizing bacteria grown on methane oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding methylketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). The product methylketones accumulated extracellularly. The rate of production of methylketones varied with the organism used for oxidation; however, the average rate of acetone, 2-butanone, 2-pentanone, and 2-hexanone production was 1.2, 1.0, 0.15, and 0.025 μmol/h per 5.0 mg of protein in cell suspensions. Primary alcohols and aldehydes were also detected in low amounts as products of n-alkane (propane and butane) oxidation, but were rapidly metabolized further by cell suspensions. The optimal conditions for in vivo methylketone formation from n-alkanes were compared in Methylococcus capsulatus (Texas strain), Methylosinus sp. (CRL-15), and Methylobacterium sp. (CRL-26). The rate of acetone and 2-butanone production was linear for the first 60 min of incubation and directly increased with cell concentration up to 10 mg of protein per ml for all three cultures tested. The optimal temperatures for the production of acetone and 2-butanone were 35°C for Methylosinus trichosporium sp. (CRL-15) and Methylobacterium sp. (CRL-26) and 40°C for Methylcoccus capsulatus (Texas). Metal-chelating agents inhibited the production of methylketones, suggesting the involvement of a metal-containing enzymatic system in the oxidation of n-alkanes to the corresponding methylketones. The soluble crude extracts derived from methane-utilizing bacteria contained an oxidized nicotinamide adenine dinucleotide-dependent dehydrogenase which catalyzed the oxidation of secondary alcohols.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Secondary Alcohols from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Over 20 new strains of methane-utilizing bacteria were isolated from lake water and soil samples. Cell suspensions of these and of other known strains of methane-utilizing bacteria oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol). The product secondary alcohols accumulated extracellularly. The rate of production of secondary alcohols varied with the organism used for oxidation. The average rate of 2-propanol, 2-butanol, 2-pentanol, and 2-hexanol production was 1.5, 1.0, 0.15, and 0.08 μmol/h per 5.0 mg of protein in cell suspensions, respectively. Secondary alcohols were slowly oxidized further to the corresponding methylketones. Primary alcohols and aldehydes were also detected in low amounts (rate of production were 0.05 to 0.08 μmol/h per 5.0 mg of protein in cell suspensions) as products of n-alkane (propane and butane) oxidation. However, primary alcohols and aldehydes were rapidly metabolized further by cell suspensions. Methanol-grown cells of methane-utilizing bacteria did not oxidize n-alkanes to their corresponding secondary alcohols, indicating that the enzymatic system required for oxidation of n-alkanes was induced only during growth on methane. The optimal conditions for in vivo secondary alcohol formation from n-alkanes were investigated in Methylosinus sp. (CRL-15). The rate of 2-propanol and 2-butanol production was linear for the 40-min incubation period and increased directly with cell protein concentration up to 12 mg/ml. The optimal temperature and pH for the production of 2-propanol and 2-butanol were 40°C and pH 7.0. Metalchelating agents inhibited the production of secondary alcohols. The activities for the hydroxylation of n-alkanes in various methylotrophic bacteria were localized in the cell-free particulate fractions precipitated by centrifugation between 10,000 and 40,000 × g. Both oxygen and reduced nicotinamide adenine dinucleotide were required for hydroxylation activity. The metal-chelating agents inhibited hydroxylation of n-alkanes by the particulate fraction, indicating the involvement of a metal-containing enzyme system in the oxidation of n-alkanes. The production of 2-propanol from the corresponding n-alkane by the particulate fraction was inhibited in the presence of methane, suggesting that the subterminal hydroxylation of n-alkanes may be catalyzed by methane monooxygenase.     

The Production of Phenylacetaldehyde from l-Phenylalanine in Tea Fermentation

Tea leaves macerated with l-phenylalanine generated rose like aroma. The gas chromatogram of the essential oil obtained from these leaves showed extremely large peak of phenylacetaldehyde. The evidence for degradation of l-phenylalanine to phenylacetaldehyde and carbon dioxide was given by the radioactive tracer experiment using l-phenylalanine-U-¹⁴C. The phenylacetaldehyde was presumed to be an intermediate product in tea fermentation from the data on the changes of the compound in tea fermentation process.     

Production of l-Phenylalanine from Starch by Analog-Resistant Mutants of Bacillus polymyxa

p-Fluorophenylalanine-resistant mutants of starch-degrading Bacillus polymyxa ATCC 842, generated by ethyl methanesulfonate mutagenesis followed by incubation with caffeine, overproduced small amounts of l-phenylalanine (l-phe) from starch. A beta-2-thienylalanine-resistant mutant (BT-7) derived from p-fluorophenylalanine mutant (C-4000 FP-4) and resistant to both p-fluorophenylalanine and beta-2-thienylalanine produced 0.5 g of l-phe and 0.15 g of l-tyrosine per liter from 10 g of starch per liter when growing in a minimal medium. trans-Cinnamic acid (CA) was also excreted by both mutants, indicating the possibility of l-phenylalanine ammonia-lyase-induced deamination of l-phe to CA. The amount of l-phe-derived CA detected in BT-7 was less compared with mutant C-4000 FP-4. CA production was induced in the parent only when l-phe was used as a sole nitrogen source. Time of CA production in the two mutants could be delayed by addition of other nitrogen sources, an indication of possible l-phenylalanine ammonia-lyase inhibition or repression. The presence of l-phenylalanine ammonia-lyase in B. polymyxa mutant C-4000 FP-4 was confirmed by assays of cell-free extracts from cells grown in starch minimal medium containing l-phe as the sole nitrogen source. Preliminary studies of the regulation of deoxy-d-arabino-heptulosonate-7-phosphate synthase and prephenate dehydratase in the wild-type strain showed that deoxy-d-arabino-heptulosonate-7-phosphate synthase was subject to feedback inhibition by l-phe, l-tyrosine, and l-tryptophan. Inhibition by each amino acid was to a similar extent singly or in combination at a 0.5 mM level of each amino acid. Prephenate dehydratase was feedback inhibited by l-phe, but not by l-tyrosine or l-tryptophan or both. In the double analog-resistant mutant BT-7, deoxy-d-arabino-heptulosonate-7-phosphate synthase had specific activity similar to that in the wild type, and the enzyme was still subject to feedback inhibition. However, prephenate dehydratase had increased specific activity and it was also insensitive to feedback inhibition by l-phe. The overproduction of aromatic amino acids by BT-7 was thought to be due, at least in part, to deregulation of feedback inhibition of prephenate dehydratase. Chorismate mutase was not subject to feedback inhibition in the wild type and was unaffected in the mutant.     

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.     

Further Studies on Production of Chloramphenicol Analogs (Corynecins) from n-Alkanes

Further studies on culture condition of Corynebacterium hydrocarboclastus were carried out for more efficient production of corynecins (chloramphenicol analogs). The productivity was affected greatly by the concentration of phosphate and stimulated effectively by organic nutrients, particularly by yeast extract. The most effective production was obtained in the presence of 8 g of yeast extract and 0.2 g of K₂HPO₄ per one liter of chemically defined medium. The maximum amount was 1.42 g equivalent of l-base (free base of chloramphenicol) per liter of the broth culture, which corresponded to 4-fold of that reported in the previous paper.

Among fractions of n-alkanes, n-heptadecane was the best carbon source for the production. The proportion among corynecins homologs varied depending on carbon number of n-alkane used; odd number alkanes gave larger amounts of corynecin II (propionyl derivative), suggesting that carbon flow in the bacterial cells functioned significantly in changing the ratios of corynecin homologs.     

Microbial Production of Xylitol from Glucose

A microbiological method is described for the production of xylitol, which is used as a sugar substitute for diabetics. A sequential fermentation process yielded 9.0 g of xylitol from 77.5 g of glucose via D-arabitol and D-xylulose. Candida guilliermondii var. soya (ATCC 20216) consumed 5.1 g of D-xylulose and produced 2.8 g of xylitol per 100 ml. Pentitol production from D-xylulose by yeasts was divided into three types: I, yeast-produced xylitol; II, yeast-produced D-arabitol; and III, yeast-produced xylitol and D-arabitol. D-Xylulose, but not glucose, was dissimilated to xylitol by yeasts under aerobic conditions.     

Production of Microbial Cells from Hydrocarbons

Hydrocarbon-assimilating yeasts and bacteria were isolated from soil and sewage. The optimal conditions of cell yield from liquid paraffine by a Torulopsis yeast and a Pseudomonas strain were studied. A Torulopsis yeast gave, in optimal condition, 70 percent cell yield on a weight conversion basis from light oil fraction. In a strain of Pseudomonas the additions of amino acids, Fe^{+ +}, Mg^{+ +} and Ca^{+ +} ions were effective for cell production. This strain showed, in optimal condition, 80 percent cell yield (wt%) from kerosene.     

Microbial Production of Theobromine from Caffeine

Production of theobromine from caffeine by caffeine-degrading bacteria was studied. We found that addition of metal ions such as Zn²⁺ to intact cells of a caffeine-degrading isolate from soil, Pseudomonas sp. No.6, resulted in a high theobromine accumulation from caffeine. We hypothesized that Zn²⁺ acts as a selective inhibitor of one of the theobromine-demethylating enzymes and further screened for theobromine-producing activities in the presence of Zn²⁺ among a number of caffeine-using microorganisms. A strain identified taxonomically as Pseudomonas putida No. 352 showed the best productivity among 973 microorganisms of stock cultures and soil isolates. Culture conditions for the production of theobromine from caffeine by P. putida No. 352 were studied. Under optimal conditions, nearly 20 g/liter of theobromine was produced from caffeine in a yield of 92%.     

Strain Improvement of Rhodotorula graminis for Production of a Novel l-Phenylalanine Ammonia-Lyase

l-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Rhodotorula rubra has been used in the commercial manufacture of l-phenylalanine from trans-cinnamic acid. In this study, R. graminis PAL was investigated. Mutant strain GX6000 was isolated after ethyl methanesulfonate mutagenesis of wild-type R. graminis GX5007 by selecting for resistance to phenylpropiolic acid, an analog of trans-cinnamic acid. Mutant strain GX6000 produced inducible PAL at levels four- to fivefold higher than had wild-type R. graminis. Furthermore, this strain had several other physiological traits that make it more commercially useful than R. rubra. For example, during fermentation, the PAL half-life was three- to fivefold longer, PAL specific activity was six to seven times higher, and PAL synthesis was significantly less inhibited by temperatures above 30 degrees C. Induction of PAL in strain GX6000 appeared to be less tightly regulated; l-leucine acted synergistically with l-phenylalanine, the physiological inducer, to increase the PAL specific activity and titer to 165 U/g (dry weight) and 3,000 U/liter, respectively, a 40% increase over the effect of l-phenylalanine alone. Strain GX6000 PAL showed significantly greater stability in bioreactors for the synthesis of l-phenylalanine, a finding that is consistent with the stability properties observed during fermentation.     

Microbial Incorporation of Fatty Acids Derived From n-Alkanes Into Glycerides and Waxes

When n-alkanes with 13 to 20 carbon atoms were fed to a Nocardia closely related to N. salmonicolor, the produced cellular triglycerides and aliphatic waxes invariably contained fatty acids with an even or an odd number of carbon atoms subject to this feature of the n-alkane substrate. Beta-oxidation and C₂ addition are both operative, as evidenced by the spectra of fatty acids incorporated into the cellular lipid components. There is no distinction in the rate of microbial incorporation of the odd-or even-numbered carbon chains. The fatty acids are apparently directly derived from the long chain n-alkanes, rather than synthesized via the classic C₂-condensation route. The alcohol component of waxes produced by the Nocardia is invariably of the same chain length as the n-alkane substrate.     

利用污泥提取液制备微生物絮凝剂

以污水厂剩余污泥作为培养基原料,经过一系列处理,探索微生物絮凝剂产生菌的最适发酵培养基配方,结果表明,污泥预处理条件以pH 12碱解条件最优,碳氮源产出量最大,补加8 g/L葡萄糖后灭菌,微生物絮凝剂产生菌LLin6可正常产絮,絮凝率达91.55%。该结果为降低微生物絮凝剂的制备成本,并实现污泥的减量化和污泥资源化利用提供了基础。     

Microbial Production of Pectin from Citrus Peel

A new method for the production of pectin from citrus peel was developed. For this purpose, a microorganism which produces a protopectin-solubilizing enzyme was isolated and identified as a variety of Trichosporon penicillatum. The most suitable conditions for the pectin production were determined as follows. Citrus (Citrus unshiu) peel was suspended in water (1:2, wt/vol), the organism was added, and fermentation proceeded over 15 to 20 h at 30°C. During the fermentation, the pectin in the peel was extracted almost completely without macerating the peel. By this method, 20 to 25 g of pectin was obtained per kg of peel. The pectin obtained was special in that it contained neutral sugar at high levels, which was determined to have a molecular weight suitable for practical applications.     

Similarities between l-Phenylalanine and m-Fluorophenylalanine as Substrates for l-Phenylalanine Ammonia-Lyase

The ability of l -phenylalanine ammonia-lyase (E.C. 4.3.1.5) to metabolize dl -m-, dl -o- and dl -p-fluorophenylalanine in Avena sativa has been examined. Although all three amino acid analogues served as substrates for this enzyme, there was a marked difference in the behavior of the meta isomer from that of the para and ortho species. The Michaelis constant for the meta analogue was similar to that obtained for the natural substrate, l -phenylalanine, but distinct from the kinetic data for the para and ortho isomers. In addition, in vivo analyses demonstrated that both l -phenylalanine and the meta-fluoro-derivative served to protect against chlorogenic acid loss, whereas previous studies have shown that the para and ortho species depressed levels of this phenolic derivative. Finally, treatment of coleoptile apices with either the meta isomer or l -phenylalanine reversed dl -p-fluorophenylalanine stimulated growth and attendant reduction in chlorogenic acid content. These findings provide further clarification of the effects of fluorophenylalanines upon l -phenylalanine ammonia-lyase mediated biosynthesis of low molecular weight phenols in Avena.     

Microbial Production of 2,3-Butylene Glycol from Cheese Whey

Six microorganisms that produced acetoin or diacetyl or both from glucose were tested for the production of 2,3-butylene glycol from lactose. Bacillus polymyxa and Streptococcus faecalis gave positive results and were tested in unmodified wheys. Cottage cheese whey was unsatisfactory, but B. polymyxa produced large amounts of the glycol in sweet whey, about 60 mmol of glycol per 100 mmol of lactose utilized. Aeration and an increased ratio of surface area to volume of whey enhanced the production of glycol. 2,3-Butylene was separated from the spent whey and from acetoin and diacetyl with a Sephadex G-10 column.     

Microbial Production of Lysine and Threonine from Whey Permeate

Extracellular accumulation of lysine and threonine was investigated in modified whey permeate by using Brevibacterium lactofermentum ATCC 21086 and Escherichia coli ATCC 21151. Whey permeate was prepared from whey by membrane ultrafiltration, and lactose was hydrolyzed by treating permeate with HCl or β-galactosidase. The highest amount of lysine (3.3 g/liter) was produced from a mixture of acid-hydrolyzed whey permeate and yeast extract (0.2%). The highest amount of threonine (3.6 g/liter) was produced from a mixture of whey permeate, (NH₄)₂SO₄ (1.4%), yeast extract (0.1%), and Na₂CO₃ (0.3%).