The flavour of many foods are formed as a result of the action of microorganisms or enzymes. Some studies have been carried out on the formation of flavour and fragrance materials using microorganism, enzymes and tissus cultures.
In this review, some of these studies are shown, and furthermore, the future contributions of biotechnology to this field are also described.

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This review was described about the microbial production of ethylene, isobutene and a saturated gaseous hydrocarbon mixture. Microbial ethylene production was studied with Penicillium digitatum IFO 9372 and a novel pathway of the ethylene biosynthesis through α -ketoglutarate was proposed. For the microbial production of isobutene, Rhodotorula minuta IFO 1102 was selected and the interesting actions of L-leucine and L-phenylalanine for the production were found. Finally, it was presented about the microbial production of a saturated gaseous hydro-carbon mixture by Rhizopus japonicus IFO 4758 and concluded that a gas mixture was produced through a chemical reaction of SR compounds and some cellular component such as squalene under aerobic conditions.
I expect this study to bring about practically feasible technology some day in future through untiring efforts.

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Over the last twenty years, some hydrocarbon-utilizing microorgainisms have been found to produce emulsifying materials, namely, biosurfactants. Biosurfactants have certain structural characteristics which are not seen in industrial synthetic surfactants. Application of biosurfactants has been attempted in a wide variety fields, including the tertiary petroleum recovery, pollution-free removing agents, hydrocarbon fermentation, industrial surfactants, and so on.
It is explained that a general outline on the current situation of research and development of these biosurfactants, according to the classification depending on the structures of their hydrophilic moieties.
And, it is explained that results of our research and development on a sophorolipid, whose derivertive has found commercial utility as a skin moisturizer.

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A novel concept 'microaqueous' (neither aqueous nor anhydrous, nonaqueous) is proposed in order to emphasize the importance of water for bio-catalysts in organic solvents and in order to cover all possible states of biocatalyst utillization in organic solvents. Optimal control of moisture content is the keystone of microaqueous Biocatalysis system because water affects not only reaction rate and selectivity of product but also stability of biocatalyst. Biocatalysts exist in microaqueous organic solvents as solubilized, fine powder suspended, immobilized in wet particle, contained in reversed micelle, solubilized by binding with polyethylene glycol, wet micribial cells suspended and so on. Microaqueous biocatalysis systems are beneficial to biochemical reactions involving water-insoluble substrates, to synthetic reactions catalyzed by reversed action of hydrolytic enzymes, to transfer reactions (e. g. transglycosylation, transpeptidylation, transesterification, transphosphatidylation, etc) by hydrolytic enzymes, and so forth. States of water in the microaqueous organic solvent is discussed. Configurations of microaqueous bioreactors and moisture sensors for both monitoring and control are also reviewed.

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Reactions of glycerol and high free fatty acid (FFA) rice bran oil were carried out with lipase immobilized on an anion exchange carrier. The esterification activity of the reactions was estimated by decrease in acid values. This activity was high when the water content of the reaction mixture was 13 %. The relationship between water content in the reaction mixture and esterification activity depended on the lipase sources and the carriers on which the lipase was immobilized. Transesterification activity was estimated by successive analysis of glyceride components in the reaction products. The yield of monoglyceride was good when both the esterification activity and transesterification were made to work in parallel. The high FFA oil used contained 35 % free fatty acid and the reaction product contained 7 15 % FFA and 30.35 % monoglyceride. The methods for controlling water content in the process of glyceride production by lipase based on differences in boiling temperatures and reactions in a triphasic system are discussed.

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Seedling growth under several different atmospheric pressures between 1 kg/cm² and 8 kg/ cm² is studied in gas mixtures composed of N₂, O₂ and CO₂. Pharbitis Nil (morning glory), Avena sativa (oats), Hibiscus esculentus (okura) and Pisum sativum (pea) are used as mate-rials. Ingeneral, hypocotyl, primary leaf, and root growth are accelerated up to 5 kg/cm² and inhibited above that pressure, especially in CO₂ containing gas mixtures. Rather little effect is observed in the cotyledon and coleoptile growth. And chlorophyll synthesis is depressed under high pressure but recovers easily when the pressure is lowered. At the end of the growing pe-riod, fresh weight is increased parallel to the increase of hypocotyl elongation, however, the dry weight is markedly decreased. And the rate of this dry weight decrease is larger in case of 5 kg/cm² than in 6 kg/cm² storage. These results seem to indicate that storage under higher pressure preserves the seed in good condition, because under higher pressure the consumption of the stored substances in the seed is smaller, and the enzyme systems keep their ability to metabolize many substances when the seed is returned to normal atmospheric pressure, as seen in the chlorophyll synthesizing system.
In experiments using 8 plant species, seeds stored under atmospheric pressure of 11 kg/cm² or 21 kg/cm² keep higher germination ability than the one stored in open air. The former seeds maintain the ability to grow normally, when they are germinated on open air experimental beds.

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The oxidation of linoleic acid catalyzed by soybean lipoxygenase (LG) I (optimum pH9.0) in a hexane-water biphasic system was studied in response to chemical modification and the use of additives. The amino groups on LG I were acylated by N-acyloxysuccinimides (1) in a phoshate buffer. The modified LG I showed enzyme activity in the biphasic system. In the unmodified form, this activity was slight due to its lack of stability. Efficiency of the modifier for bringing about activity increased with CN (carbon nunber of acyl group) in (1). On the other hand, stability decreased with increasing CN in the modifier. The addition of BSA modified by (1) (CN=6 and 8) also elicited activity in the biphasic system, while additives such as BSA, casein and NaCl had no significant effect.
Based on these observations, a mechanism for the occurrence of LG activity in the biphasic system is proposed and discussed.

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Sudies were carried out to evaluate the surface active properties of rhamnolipid B [2-O- (2-O- α-decenoyl-α-L-rhamnopyranosyl) - α -L-rhamnopyranosyl-β-hydroxydecanoyl- β-hydroxydecanoic acid] and its precursor A as microbial biosurfactants produced by the hydro-carbon-assimilating bacterium, Pseudomonas aeruginosa BOP 100. The micelle formation of the sodium salts of rhamnolipids was confirmed by the detection of critical micelle concentration s (cmc) of A : 6.22 × 10^-5, B : 1.50 × 10^-4M (fluorescence intensity of ANS) and by micellar weight determinations of A : 38000, B : 7000 (light scattering), in spite of the bulky and complicated structures of the micelles, consisting of alternate hydrophobic and hydrophilic portions in contrast to synthetic surfactants. The γ cmc values of the sodium salts of A and B were 28 mN/m, while their interfacial tension was 0.2 and 3.2 mN/m at the interface between 0.1% solution and kerosene. Moreover, highly efficient gross effects were also noted particularly in dispersing, emulsifying, foaming and penetrating action. From these findings rhamnolipids may be concluded to have large surface activity and polyfunctionality on the basis of the amphiphiles as membranous constituents.

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From Bacillus subtilis ATCC No. 21813, barley β-glucanase and α-amylase were produced simultaneously in a submerged culture.
To obtain barley β-glucanase with less α-amylase the culture process using this Bacillus sp. was investigated.
The addition of (NH₄)₂HPO₄ or FeSO₄ to the broth filtrate depressed α-amylase activity while maintaining that of β-glucanase.
The addition of both these compounds to the culture medium gave the same results in the culture process. Essentially the same results were obtained using other Bacillus sp.

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From out of 76 types of culture strains, wax-ester-accumulating microorganisms were screened using oleic acid as the sole carbon source.
Acinetobacter calcoaceticus IAM 12088 was found to be microorganism accumulating the most wax. It produced 4.9g/L of wax esters from 30g/L of oleic acid. These wax esters consisted mainly of the fatty acid and alcohol having C_{18 : 1}, C_{16 : 1} and C_{14 : 1}, units. C_{16 : 1} and C_{14 : 1} fatty acids may possibly have formed from oleic acid by β-oxidation. After reduction of the C_{18 : 1}, C_{16 : 1} and C_{14 : 1} fatty acids to the corresponding fatty alcohols, ester formation between fatty acids and fatty alcohols took place. Wax esters were also produced from triglycerides such as palm and mink oil as sole carbon sources.
The mutant having decreased capacity to degrade wax esters was isolated by NTG from the selected strains. The mutant accumulated wax esters in amounts exceeding 3 times that of parental strain, the yield being 9.7g/L and conversion rate, 48.5% from 20g/L of oleic acid. Supplementation of oleic acid and corn steep liquor during cultivation gave the best yield of 30.5 g/L. The maximum conversion rate of oleic acid to wax esters was 53.5%.

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The stereoselective hydrolysis of the long-chained substrate (p-nitrophenyl N-dodecanoyl-D (L) -phenylalaninate, D (L) -S₁₂) has been found to be controlled by addition of unsaturated fatty acids or micelles in the artificial membrane systems. The noteworthy aspects are as follows : (a) Methyl oleate enhanced the catalytic efficiency (reflected in the second-order rate constant, ka, obsd) for the hydrolysis of D(L)-S₁₂ in the catalytic system of N-benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine (Z-PheHisLeu) and ditetradecyldimethylammonium bromide (2 C₁₄Br) along with the elevation of phase transition temperature. On the other hand, (b)γ-irradiated methyl linoleate reduced the catalytic efficiency of Z-PheHisLeu with time course. This would be attributed to the autoxidation of γ-irradiated methyl linoleate; (c) the high enantioselectivity (k^L_aobsd/k^D_aobsd =28) of S¹² with Z-PheHisLeu in the membrane system of 2 C₁₄ Br (1mM) was elevated dramatically by addition of micelles (hexadecylbenzyldimethylammonium chloride, 7 mM) and attained to enantiomer rate ratio (k^L_aobsd/k^D_aobsd =67) at room temperature.

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The hydrolysis of borage seed oil containing γ-linolenic acid (Δ6, 9, 12-octadecatrienoic acid) was investigated using two kinds of microbial lipases.
The Candida cylindracea lipase easily hydrolized α-linolenate (Δ9, 12, 15-octadecatrienoate) present in linseed oil but not so easily γ-linolenate in borage seed oil. Far example, when borage seed oil was hydrolized by the C. cylindracea lipase, γ-linolenate accumulated in the unhydrolized glyceride residue. This difference for affecting hydrolysis is likely due to the specificity of this lipase toward γ-linolenate containing Δ6 unsaturation. In contrast, using the Chromobacterium viscosum lipase, there was no such accumulation of γ-linolenate. The difference in microbial origin of these lipases may possibly account for these findings.

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To standardize the assay method of lipase with the emulsified substrate, some important factors affecting lipase activity were investigated.
The coefficient of variation (C.V.) in lipase activity by lot of olive oil were different among origins of lipase. C.V. were less than 10% in the case of assaying 10 times in one assay (within-run) and its value in duplicate in twice at regular intervals (between-run) was approximately 12%.
The suitable agitation number to get the highest lipase activity varied with origins of lipase. The agitation number to be suitable for any origin of lipase was 15, 000 rpm to 16, 000 rpm.
To keep emulsified substrate stable, addition of the mixture of PVA 117 and PVA 205 was better than when either is added alone.

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