Microbial Conversion of Petro-sulfur Compounds

In order to enhance the efficiency converting dibenzothiophene (DBT) into water-soluble compounds by DBT-utilizing bacteria, the following subjects were studied: comparison of activities of isolated strains, their mixed cultivation, DBT dosing, nitrogen sources, substrate concentration, pH, temperature, and oxygen supply. Light oil solution of DBT (5%) was used as substrate. The conversion efficiency was calculated by the amount of sulfur accumulated in the aqueous layer of culture broth.

The conversion ratio of 40% was attained by the mixed culture of Pseudomonas abikonensis and Ps. jianii under the following conditions; medium, DBT 4.6 g dissolved in 87.8 g of light oil, meat extract 4.0 g, Na₂HPO₄·12H₂O 9.5 g, KH₂PO₄ 1.4 g, MgCl₂·6H₂O 0.2 g, distilled water 1000 ml; pH, 6.9 to 7.3 (m/30 phosphate buffer); oxygen supply, 50 ml broth 500 ml flask, shake culture at 220 rpm; inoculum size, 10%; temperature, 28°C; and period of cultivation, 3 days.     

Microbial Conversion of Petro-sulfur Compounds

Five substances, water soluble organic sulfur compounds, produced from dibenzothiophene by such bacteria as Pseudomonas jianii or Ps. abikonensis were isolated from culture broth. Three products of them were identified as 3-hydroxy-2-formyl-benzothiophene, dibenzothiophene-5-oxide and 3-oxo-2[3′-hydroxy-thionaphthenyl-(2)-methylene]dihydrothionaphthene respectively.     

Microbial Conversion Products of Leptomycin B

Leptomycin B (LMB), a secondary metabolite produced by Streptomyces sp. strain ATS 1287, with known antifungal and antitumor effects, inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 regulatory protein Rev and exhibits significant antiproliferative activity. Since LMB itself turned out to be distinctly cytotoxic, a bioconversion screening with a selected set of 29 bacterial and 72 fungal strains was performed in order to obtain metabolites of LMB with reduced antiproliferative effects. Several derivatives of LMB, more polar than the parent compound and produced in yields of >5%, were detected. Liquid chromatography-mass spectroscopy analysis indicated the type of bioconversion. Fermentations (1-liter scale) of those strains with high rates of transformation were suitable for isolation and characterization of the most prominent metabolites. Thus, bioconversion of LMB with Aspergillus flavus ATCC 9170 and Emericella unguis ATCC 13431 served for isolation of the novel derivatives 26-hydroxy-LMB (30% was the concentration of the metabolite [with respect to LMB] used for bioconversion) and LMB-24-glutaminamide (90%), respectively. Streptomyces rimosus ATCC 28893 converted LMB into 4,11-dihydroxy-LMB (13%) and 2,3-dihydro-LMB (55%). Although the antiproliferative effects of the LMB metabolites could be reduced through microbial conversion, none of these metabolites inhibited the nuclear export of Rev better than LMB itself.     

Microbial Transformation of Nitroaromatic Compounds in Sewage Effluent

The transformation of mono- and dinitroaromatic compounds was measured in sewage effluent maintained under aerobic or anaerobic conditions. Most of the nitrobenzene, 3- and 4-nitrobenzoic acids, and 3- and 4-nitrotoluenes and much of the 1,2- and 1,3-dinitrobenzenes disappeared both in the presence and absence of oxygen. Under anaerobiosis, 2,6-dinitrotoluene and 3,5-dinitrobenzoic acid disappeared slowly, but no loss was evident in 28 days in aerated sewage. Aromatic amines did not accumulate during the aerobic decomposition of the mononitro compounds. They did appear in nonsterile, but not in sterile, sewage incubated aerobically with the dinitro compounds and anaerobically with all the chemicals. Analysis by gas chromatography and combined gas chromatography-mass spectrometry showed that aniline was formed from nitrobenzene, toluidine was formed from 3- and 4-nitrotoluenes, and aminobenzoic acid was formed from 3- and 4-nitrobenzoic acids under anaerobiosis, and that nitroaniline was formed from 1,2- and 1,3-dinitrobenzenes, aminonitrotoluene resulted from 2,6-dinitrotoluene, and aminonitrobenzoic acid was a product of 3,5-dinitrobenzoic acid under both conditions. The isomeric forms of the metabolites were not established. Aniline, 4-toluidine, and 4-aminobenzoic acid added to sewage disappeared from aerated nonsterile, but not from sterile, sewage or sewage in the absence of oxygen. 2-Nitroaniline, 2-amino-3-nitrotoluene, and 2-amino-5-nitrobenzoic acid added to sewage persisted for at least 60 days in aerobic or anaerobic conditions. Gas chromatographic and gas chromatographic-mass spectrometric analyses demonstrated that acetanilide and 2-methylquinoline were formed from aniline, 4-methylformanilide and 4-methylacetanilide were formed from 4-toluidine, 2-methylbenzimidazole was a product of 2-nitroaniline, and unidentified benzimidazoles were formed from 2-amino-3-nitrotoluene in the absence of oxygen, and that 2-nitroacetanilide and 2-methyl-6-nitroacetanilide were formed from 2-nitroaniline and 2-amino-3-nitrotoluene, respectively, in the presence or absence of oxygen. It is suggested that the transformations of widely used nitroaromatic compounds should be further studied because of the persistence and possible toxicity of products of their metabolism.     

微生物降解硝基芳香族化合物(NAC)的研究进展

硝基芳香族化合物是非常重要的有机化工原料,也是难降解有机污染物之一。相对于传统去除法,利用微生物矿化或非特异性的转化,使硝基芳香族化合物成为生物地化循环的一部分,从而降低对环境污染的修复手段更具有可持续性。本文综述了降解硝基芳香族化合物的微生物资源及其降解途径、降解机理、相关修复方式等的研究进展。     

Microbial Conversion of alpha-Ionone, alpha-Methylionone, and alpha-Isomethylionone

alpha-Ionone, alpha-methylionone, and alpha-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from alpha-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-alpha-ionone, trans-3-hydroxy-alpha-ionone, and 3-oxo-alpha-ionone. 2,3-Dehydro-alpha-ionone, 3,4-dehydro-beta-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from alpha-methylionone and alpha-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for alpha-ionones and elucidation of stereochemical features of the bioconversion.     

微生物酶法转化生产L-肉碱的研究进展

L -肉碱作为一种新型的营养强化剂和临床药物 ,广泛应用于医疗、保健、食品等领域。L- 肉碱的生产方法有化学合成、微生物发酵、微生物酶法转化等 ,其中微生物酶法转化被认为是一种最经济且最有前途的方法。就 3种酶法转化 (DL -肉碱衍生物的酶法拆分、巴豆甜菜碱的酶法转化、D- 肉碱的酶法转化 )的微生物产酶菌株、产酶条件和酶法转化的最适条件作一概述。     

Adaptation of Aquatic Microbial Communities to Quaternary Ammonium Compounds

The effects of long-chain (C₁₂ to C₁₈) quaternary ammonium compounds (QACs) on the density, heterotrophic activity, and biodegradation capabilities of heterotrophic bacteria were examined in situ in a lake ecosystem. Monoalkyl and dialkyl substituted QACs were tested over a range of concentrations (0.001 to 10 mg/liter) in both acute (3 h) and chronic (21 day) exposures. In general, none of the QACs tested had significant adverse effects on bacterial densities in either acute or chronic studies. However, significant decreases in bacterial heterotrophic activity were noted in acute studies at QAC concentrations from 0.1 to 10 mg/liter. Chronic exposure of lake microbial communities to a specific monoalkyl QAC resulted in an adaptive response and recovery of heterotrophic activity. No-observable-effect level in the adapted populations was >10 mg/liter. Chronic exposure also resulted in significant increases in the number and activity of bacteria capable of biodegrading the material. The increase in biodegradation capability was observed at low (microgram per liter) concentrations which are approximately the same as realistic environmental levels. In general, our studies indicated that exposure of lake microbial communities to QACs results in the development of adapted communities which are less sensitive to potential toxic effects and more active in the biodegradation of these materials.     

Microbial Conversion of Tall Oil Sterols to C19 Steroids

Mycobacterium sp. NRRL B-3683 converted tall oil sterols to C₁₉ steroids as efficiently as it converted soybean sterols.     

Degradation of Nucleic Acids and Related Compounds by Microbial Enzymes

The culture filtrate of a strain of Bacillus subtilis decomposed ribonucleic acid into 5′-nucleotides and into other intermediates which released orthophosphate by an arsenate-resistant phosphatase. Under the best conditions examined in these experiments, about 50 per cent of ribonucleic acid was converted into 5′-nucleotides.

The culture filtrate of a strain of Bacillus brevis showed slight activities of ribonuclease and/or phosphodiesterase which produced 5′-nucleotides from ribonucleic acid, but showed predominant activity of 5′-adenylic acid degrading phosphatase.     

Assessment of Microbial Fouling in an Ocean Thermal Energy Conversion Experiment

A project to investigate biofouling, under conditions relevant to ocean thermal energy conversion heat exchangers, was conducted during July through September 1977 at a site about 13 km north of St. Croix (U.S. Virgin Islands). Seawater was drawn from a depth of 20 m, within the surface mixed layer, through aluminum pipes (2.6 m long, 2.5-cm internal diameter) at flow velocities of about 0.9 and 1.8 m/s. The temperature of the seawater entering the mock heat exchanger units was between 27.8 and 28.6°C. After about 10 weeks of exposure to seawater, when their thermal conductivity was reported to be significantly impaired, the pipes were assayed for the accumulation of biological material on their inner surfaces. The extent of biofouling was very low and independent of flow velocity. Bacterial populations, determined from plate counts, were about 10⁷ cells per cm². The ranges of mean areal densities for other biological components were: organic carbon, 18 to 27 μg/cm²; organic nitrogen, 1.5 to 3.0 μg/cm²; adenosine 5′-triphosphate, 4 to 28 ng/cm²; carbohydrate (as glucose in the phenol assay), 3.8 to 7.0 μg/cm²; chlorophyll a, 0.2 to 0.8 ng/cm². It was estimated from the adenosine 5′-triphosphate and nitrogen contents that the layer of live bacteria present after 10 weeks was only of the order of 1μm thick. The C/N ratio of the biological material suggested the presence of extracellular polysaccharidic material. Such compounds, because of their water-retaining capacities, could account for the related increase in thermal resistance associated with the pipes. This possibility merits further investigation, but the current results emphasize the minor degree of biofouling which is likely to be permissible in ocean thermal energy conversion heat exchangers.     

Degradation of Phenol Compounds by Microbial Communities of the Amur Estuary

The microbiological transformation of various phenol compounds and their origin in the mixing zone of the Amur Estuary is considered. The results of the model experiments on the decomposition and transformation of phenol compounds by microbial communities and bacteria are reported. Self-depuration of the Amur Estuary depends on the mechanisms of the transformation of aromatic compounds in the presence of cosubstrates, the enzymatic activity of microbial communities, and the temperature. In winter, the ecological risk of pollution by aromatic compounds increases.     

宏基因组克隆--微生物活性物质筛选的新途径

在现有技术条件下自然界存在的微生物95％以上未能培养,采用传统的分离培养筛选的途径寻找新的微生物生物活性物质受到局限;宏基因组是特定小生境中全部微小生物遗传物质的总和,直接抽提环境样品中的总DNA,利用适宜的载体克隆到替代宿主细胞中构建宏基因组文库,通过外源基因赋予宿主细胞的新性状或基于某些已知DNA序列筛选,寻找新的生物活性物质或基因,极大地扩展了微生物资源的利用空间,增加了获得新的生物活性物质的机会。     

Degradation of Nucleic Acids and Their Related Compounds by Microbial Enzymes

The optimum pH of the DNA-depolymerase produced by Aspergillus quercinus was found to be about 8.5 and maximal formation of the enzyme in the culture medium was observed at the 96th hour. The culture filtrate of Aspergillus quercinus hydrolyzed DNA into 5′-deoxy-mononucleotides at a pH range higher than 6.0. Each deoxymononucleotide was isolated as crystals in good yield from an enzymatic digest of DNA and characterized spectfophotometri-cally, enzymatically and by determination of its nitrogen and phosphorus composition. 5′-Deoxyinosinic acid was obtained by hydrolysis of DNA with Streptomyces aureus. 5′-Deoxyribo-tides of hypoxanthine and guanine possessed an attractive taste very similar to that of 5′-ino-sinic and 5′-guanylic acids.     

Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

Attempts were made to form 5′-mononucleotides from non-proliferating cells of various kinds of yeasts. It was found that yeasts were devided into four groups according to their ability to excrete compounds absorbing at 260 mμ, the first of which excreted UV-absorbing materials at the acid pH (I), the second at the alkaline pH (II), the third both at the acid and alkaline pHs (III), and the fourth showed weak ability to excrete UV-absorbing materials at both pHs (IV). In general, 3′-mononucleotides were excreted at the acid pH and 5′-mononucleotides at the alkaline pH. Rhodotorula pallida, however, excreted 5′-mononucleotides both at the acid and alkaline pHs.     

Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

The distribution in microorganisms of extracellular enzymes which degrade RNA into 5′-mononucleotides was studied. The degradation products of RNA were determined by using 5′-nucleotidase and adenosine deaminase.

It was found that the enzymes were produced by various microorganisms belonging to Streptomyces, Bacillus, Fungi imperfecta such as Fusarium, Helminthosporium, etc., and Ascomycetes such as Neurospora, Glomerella, Aspergillus, etc.     

Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

Aspergillus quercinus (IFO 4363) was selected as the most suitable strain to produce 5′-mononucleotides from RNA among several species of Aspergillus which produced enzymes capable of degrading RNA into 5′-mononucleotides.

Aspergillus quercinus produced two kinds of RNA-depolymerases (designated as RNA-deploymerase I and II), phosphodiesterase, phosphomonoesterase and adenylic deaminase in the culture medium. The optimum pH of each enzyme was found to be about 4.5, 7.0, 5.0, 6.0 and 5.5, respectively. Maximal production of these enzymes in the culture medium occurred at 96, 96, 216, 168 and 264 hour culture, respectively. The culture filtrate of Aspergillus quercinus degraded RNA into 3′-mononucleotides at the pH lower than 6.0, into 5′-mono-nucleotides at the pH higher than 8.5 and into both mononucleotides at the pH range between 6.0 and 8.5. 5′-Inosinic acid was prepared from RNA by using the extra- and intracellular enzymes of Aspergillus quercinus.     

Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

Seven strains of microorganisms selected by the previous screening tests were further compared on their ability to produce extracellular enzyme systems capable of degrading RNA into 5′-ribonucleotides. As a result, two strains of Streptomyces were finally concluded to be most preferable. When these two were applied, the rate of 5′-nucleotide production reached up to 70%.

Bacillus subtilis was outstanding in its activity to degrade RNA, but its PDase activity producing 5′-nucleotides from RNA was found to be lower than those of Streptomyces strains. A pathway involving 3′- and 5′-nucleotides as intermediates was proposed for the degradation of RNA by the Bacillus enzyme system. The activity of RNA-degrading enzyme system of Bacillus subtilis contained in the supernatant of culture fluid was found to be lost at 700°C but remained to certain extent at 100°C, a possible mechanism for the phenomenon being discussed. Usability of the Bacillus enzyme system in the practical production of 5′-nucleotides under the condition of high RNA concentration was discussed.