Studies on the Utilization of Hydrocarbons by Microorganisms

During the course of an investigation of the microbial assimilation of aromatic hydrocarbons, several strains were found to produce a large amount of cumic acid from p-cymene.

Five strains, S449B1, B2, B3, B4 and B6, were isolated from soil with the aromatic hydrocarbon substrates. They all assimilated both p-cymene and cumene. The strain S449B3 grew also on p-xylene, and S449B6 on p-xylene, toluene, and ethylbenzene.

They were all shown to be capable of producing an ultraviolet-absorbing substance from p-cymene. This substance was isolated in crystalline form and identified as cumic acid by infrared absorption spectrum and other observations.

The superior strain, S449B6, produced the acid as much as 1000 mg/1 in shaking culture at 30°C after 24 hours. The yields were increased up to approximately 1700 mg/1 after further investigations. Addition of calcium carbonate and considerable agitation were favorable conditions for the acid production.

The taxonomical studies of these strains were carried out, and they were all identified as closely resembling Pseudomonas desmolytica.     

Microbial Oxidation of α-Methylstyrene and β-Methylstyrene

An unidentified bacterial strain S107B1, isolated from soil by use of isopropylbenzene as a carbon source, was shown to bring about oxidation of α-methylstyrene and β-methylstyrene,

One of the oxidation products produced from α-methylstyrene was identified as the new compound, (—)-cis-23-dihydroxy-1-isopropenyl-6-cyclohexene.

The same strain S107B1 also oxidized β-methylstyrene and produced 3-phenylpropionaldehyde and benzoic acid.

From these results, the existence of reductive step for the aerobic degradation of these aromatic hydrocarbons by this strain was made clear. The initial attack on these aromatic hydrocarbons and a cyclohexenediol compound formed from α-methylstyrene were discussed.     

Bacillus halotolerans strain LYSX1-induced systemic resistance against the root-knot nematode Meloidogyne javanica in tomato

Purpose

Determination of the nematicidal potential and mode of action of bacteria isolated from tobacco rhizosphere soil against the root-knot nematode Meloidogyne javanica in tomato plants.

Methods

Antagonistic bacteria were isolated from rhizosphere soil of tobacco infested with root-knot nematodes. Culture filtrate was used to examine nematicidal activity and ovicidal action of bacterial strains. Biocontrol of M. javanica and growth of treated tomato plants were assessed in pot experiments. To clarify whether secondary metabolites of bacteria in tomato roots induced systemic resistance to M. javanica, bacterial culture supernatants and second-stage juvenile nematodes were applied to spatially separated tomato roots using a split-root system. Bacterial strains were identified by 16S rDNA and gyrB gene sequencing and phylogenetic analysis.

Results

Of the 15 bacterial strains isolated, four (LYSX1, LYSX2, LYSX3, and LYSX4) demonstrated nematicidal activity against second-stage juveniles of M. javanica, and strain LYSX1 showed the greatest antagonistic activity; there was dose-dependent variability in nematicidal activity and inhibition of egg mass hatching by strain LYSX1. In vivo application of LYSX1 to tomato seedlings decreased the number of egg masses and galls and increased the root and shoot fresh weight. Treatment of half of the split-root system with LYSX1 reduced nematode penetration to the other half by 41.64%. Strain LYSX1 was identified as Bacillus halotolerans.

Conclusion

Bacillus halotolerans LYSX1 is a potential microbe for the sustainable biocontrol of root-knot nematodes through induced systemic resistance in tomato.

     

Identification and Antimicrobial Activity of Phenylacetic Acid Produced by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> Isolated from Fermented Soybean,Chungkook-Jang

A bacterial strain, B65-1, which showed strong antimicrobial activity, was isolated from Chungkook-Jang, a traditional Korean fermented-soybean food with antimicrobial properties. Based on carbon utilization pattern and partial 16S rRNA sequence analysis, the B65-1 strain was identified as Bacillus licheniformis. An antibiotic compound, active against bacteria and yeast such as Staphylococcus aureus, Escherichia coli, and Candida albicans, was isolated by various chromatographic procedures from culture filtrates of B. licheniformis B65-1. The purified antibiotic was identified to be phenylacetic acid, with the molecular formula C₈H₈O₂ by analyses of EI-MS and NMR. The phenylacetic acid was detected in fermented soybean made with the strain B65-1 as a starter, but was not present in extracts of nonfermented soybean. Our results indicated that the phenylacetic acid produced by B. licheniformis during fermentation of soybean is one of the main compounds of antimicrobial activity of Chungkook-Jang.     

Isolation and Identification of a Microorganism which Produces Non Streptomyces Pepsin Inhibitor and N-Diazoacetyl-dl-norleucine Methylester Sensitive Acid Proteases

Non pepsin inhibitor (S–PI) and diazoacetyl-dl-norleucine methylester (DAN) sensitive acid proteases producing microorganism was isolated from farm soil of Osaka Prefecture.

The isolated strain was identified as Scytalidium lignicolum M–133. When it was aerobically grown on a medium consisting of glucose 5%, meat extract 1.5%, yeast extract 0.1%, KH₂PO₄ 0.2%, MgSO₄·7H₂O 0.05% at pH 3.5 and 25°C, the strain produced two acid proteases, A and B, in the culture broth.

The acid proteases A and B were not at all inactivated by S–PI and DAN. These acid proteases were expected to be a new type of acid protease from the viewpoint of the active site.     

Biodegradation of endosulfan and endosulfan sulfate by <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> strain C8B in broth medium

Endosulfan is one of the most widely used wide spectrum cyclodiene organochlorine insecticide. In environment, endosulfan can undergo either oxidation or hydrolysis reaction to form endosulfan sulfate and endosulfan diol respectively. Endosulfan sulfate is as toxic and as persistent as its parent isomers. In the present study, endosulfan degrading bacteria were isolated from soil through selective enrichment technique using sulfur free medium with endosulfan as sole sulfur source. Out of the 8 isolated bacterial strains, strain C8B was found to be the most efficient endosulfan degrader, degrading 94.12% α-endosulfan and 84.52% β-endosulfan. The bacterial strain was identified as Achromobacter xylosoxidans strain C8B on the basis of 16S rDNA sequence similarity. Achromobacter xylosoxidans strain C8B was also found to degrade 80.10% endosulfan sulfate using it as sulfur source. No known metabolites were found to be formed in the culture media during the entire course of degradation. Besides, the bacterial strain was found to degrade all the known endosulfan metabolites. There was marked increase in the quantity of released CO₂ from the culture media with endosulfan as sulfur source as compared to MgSO₄ suggesting that the bacterial strain, Achromobacter xylosoxidans strain C8B probably degraded endosulfan completely through the formation of endosulfan ether.     

Antibiotic Complex SP-351 Produced by a Psychrophile,Streptomyces sp. No. 351

In the course of a screening study for antibiotics using psychrophilic microorganisms a water-insoluble antibiotic complex, SP–351, was found in the culture filtrate of a psychrophilic actinomycete, strain No. 351. This active principle was isolated and characterized as a cyclicpolylactone antibiotic. The SP–351-producing strain was classified as a facultative psychrophile and identified as Streptomyces phaeochromogenes.

The main components of the antibiotic complex SP–351 changed with the composition of the culture medium but not with culture temperatures. Component A was exclusively produced in a medium composed of roasted soybean powder and glycerol; components B and C in a medium composed of soybean, glycerol and potassium nitrate; and components A and D in a synthetic medium containing a hydrocarbon, alcohol or ester as the sole carbon source. Maximum production of SP–351 from n-paraffin and methyl acetate was 10 and 15 mcg/ml, respectively.

SP–351 showed strong antibacterial activity against Gram-positive bacteria and acid-fast bacteria at 0.1~0.3 mcg/ml concentrations.     

Isolation,Identification and Substrate Assimilation Specificity of Some Aromatic Hydrocarbon Utilizing Bacteria

As a result of screening isoalkyl or isoalkenyl substituted aromatic hydrocarbon assimilating microorganisms, 19 strains of isopropylbenzene assimilating bacteria were isolated. Thirteen of these strains were found to grow on α-methylstyrene and all 4 strains tested were also found to grow on isobutylbenzene.

Among them, 2 strains (S107B1 and S182BI) were selected for further study and were identified as Ps. convexa and Ps. ovalis, respectively.

Furthermore, some examined aromatic hydrocarbon utilizing bacteria were classified into two groups by differences in substrate assimilation specificity.     

桑树内生拮抗菌的分离鉴定及其对桑断枝烂叶病的生防初探

【目的】本研究从健康桑树茎中分离筛选对桑断枝烂叶病菌具显著拮抗作用的内生细菌,为该病生物防治奠定研究基础。【方法】采用组织培养法分离桑树内生菌,抑菌圈法和平板对峙法筛选抑菌活性稳定的内生拮抗菌;根据形态学、生理生化特征检测和基于16SrDNA、gyrA和gyrB基因的系统发育分析对拮抗菌进行菌种鉴定;利用抑菌圈法测定拮抗菌株活性发酵液热稳定性,菌丝生长速率法检测活性发酵液抑菌谱;并通过观察拮抗菌对桑断枝烂叶病菌BoeremiaexiguaGXH1菌株生长及菌丝形态的影响,扩增抑菌活性物质合成关键基因,以及采用酸沉淀法提取拮抗菌株脂肽类化合物并进行高效液相色谱串联质谱分析(LC-MS),初步探究可能的抑菌机制。【结果】从健康桑树茎中共分离获得17株桑树内生细菌,并从中筛选获得一株对桑断枝烂叶病菌B.exiguaGXH1有稳定拮抗作用的桑树内生细菌NPJ13菌株。该菌株形态学、生理生化特征与芽孢杆菌属一致,基于16SrDNA、gyrA和gyrB基因序列的系统发育分析结果显示该菌株与贝莱斯芽孢杆菌(Bacillusvelezensis)的亲缘关系最近,且处于系统发育树的最小分枝,故将NPJ13菌株鉴定为贝莱斯芽孢杆菌,命名为B. velezensis NPJ13。NPJ13菌株对灰霉病菌SWU5、核地杖菌SXSG-5、核盘菌PZ-2及烟草疫霉SWU20等12种病原真菌具有不同程度的拮抗作用,其活性发酵液具有较好的热稳定性。NPJ13菌株会导致桑断枝烂叶病菌GXH1菌丝发生扭曲、膨大、透明度增加、断裂等畸变现象;基因检测结果显示NPJ13菌株基因组中具有PKSI、NRPS、Sfp、ItuD、Srfc等5种抑菌活性物质合成关键基因,LC-MS检测结果表明菌株NPJ13脂肽类粗提物中含有表面活性素和伊枯草菌素。【结论】本研究分离筛选获得一株对桑断枝烂叶病菌具有显著拮抗作用的桑树内生细菌B. velezensis NPJ13菌株,为桑断枝烂叶病的生物防治提供了候选菌株。     

Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.     

Formation of Desthiobiotin from Oleic Acid by Brevibacterium sp.

Microbial formation of biotin-vitamers from oleic acid was investigated. Many strains of bacteria which were able to utilize oleic acid as a sole carbon source were isolated from soils and other natural materials. Among these bacteria, some strains formed a biotin-vitamer from oleic acid in the culture broth during the cultivation. The vitamer was purified from the culture broth of strain No. 23, and identified as desthiobiotin by chromatographical and biological methods.

From the results of investigation on the taxonomical characteristics, the bacterial strain No. 23 was assumed to be Brevibacterium sp.     

Genomic Analysis and Comparative Hexavalent Chromium Reduction Potential of Predominant Bacillus species Isolated from Chromite Mine Soil

Ten predominant bacteria (CSB 1-10), isolated from chromite mine soil of Sukinda, Odisha, were characterized by means of biochemical and 16S rRNA gene sequencing. All of the bacterial isolates were Gram-positive, spore-forming, and motile rods with diameter ranging from 1.57–2.79 μm and identified as Bacillus species. Based on 16S rRNA gene sequencing and phylogenetic tree construction, 10 Bacillus species were grouped into two clusters: Bacillus subtilis cluster with four species (two of B. amyloliquefaciens, and one each of B. tequilensis and B. mojavensis); and Bacillus cereus cluster containing six species of B. cereus. Secondary rRNA structure predicted for all 10 bacteria using 16S rRNA sequences revealed some degree of genetic variations among the species. Among the isolated bacteria, CSB-9 was found to be the most efficient chromate reducing strain (0.8 × 10^?4 mg mg^?1 h^?1) in comparison to the others. Chromate reduction of the bacteria was associated with the contribution of extracellular enzyme production, and highest enzyme activity (0.9 ± 0.09 U mL^?1) was observed in CSB-9 (B. amyloliquefaciens). The present study revealed the dominance of Bacillus species in the chromite mine soil and their potential for bioremediation of hexavalent chromium from the polluted environments.     

三七皂苷类自毒物质降解细菌分离及其降解特性

三七是我国的名贵药材,但由于连作障碍发生严重,因此土壤中自毒物质的积累成为导致三七连作障碍发生的主要原因之一。生物降解土壤中的自毒物质是缓解连作障碍的有效措施,为筛选并利用降解菌使土壤中皂苷类自毒物质快速消减,该研究以皂苷类自毒物质为筛选靶标,采用富集和驯化策略,从连作三七根际土壤中分离、筛选三七皂苷类自毒物质降解细菌,结合16S rRNA基因测序对高活性菌株进行分类鉴定,并对筛选得到的高活性菌株SC3的降解特性进行了研究。结果表明:(1)从三七根际土壤中成功分离出8株潜在自毒物质降解细菌,初筛评价结果显示SC3菌株对三七总皂苷的降解率最高,达87.42%。(2)通过16S rRNA基因序列分析,编号SC3的高活性菌株被鉴定为寡养单胞菌属(Stenotrophomonas)细菌。(3)在相同培养条件下,菌株SC3对单体皂苷Rb₁的降解效果强于对Rg₁的降解。(4)在液体培养条件下,底物浓度、接种量和培养温度均会显著影响SC3菌株对单体皂苷Rb₁的降解效果。综上表明,采用富集和驯化策略可以有效筛选自毒物质降解细菌,SC3菌株具有消除连作土壤中皂苷类自毒物质的潜力。该研究结果为连作土壤修复提供了生物资源,并为今后深入研究皂苷降解机制提供了理论依据。     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazolecarboxamide Riboside by Escherichia coli

The acculnulation of 5 (4) -amino-4 (5) -imidazolecarboxamide riboside (AICA-R) in the culture medium of sulfonamide-inhibited Escherichia coli, and E. coli-like bacteria was studied. E. coli strain Band 32 strains of E. coli-like bacteria accumulated more than 50 μmoles of AICA-R in test tube scale experiments, and one of E. coli-like bacteria accumulated 358 μmoles. E. coli B-96 (purine-requiring mutant) had ability to accumulate AICA-R in the glucose-salt medium containing purine bases, especially xanthine. The addition of glycine alone or together with glutamic acid to the glucose-salt medium increased the accumulation of AICA-R by sulfadiazine-inhibited E. coli strain B. The accumulation was considerably increased by the addition of polypeptone or casein hydrolysate.

AICA-R accumulated during sulfadiazine bacteriostasis of E. coli strain B was purified and crystallized according to the procedure of Greenberg and Spilman, and light amber colored crystals were obtained.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

1. Suitable agar plate media were selected for isolation of nucleotide producing strains, by salvage synthesis, from natural sources. Since this agar medium contains a high concentration of phosphates, manganese and glucose, it is specific for these bacteria.

2. With this plate medium, 113 bacterial strains accumulating 5′inosinic acid (IMP) or IMP-like substances were isolated effectively from feces of a variety of birds and mammals and from soils.

Some of the strains isolated were recognized to accumulate other nucleotides, purine bases and sugars, such as guanine nucleotides, XMP, xanthine, ribulose or xylnlose, with or without hypoxanthine in the media.

3. Five strains of IMP accumulating bacteria were identified; two were classified as Brevibacteriurm, two as Corynebacterium and one as Arthrobacterium species by taxonomical studies. But their characteristics did not completely coincide with those of bacteria described in Bergey’s manual.

4. One of the IMP producing bacteria isolated, culture No. 21–26, actually consisted of two separate strains, namely No. 21–26–101 and No. 21–26–102. The highest production of IMP or guanine nucleotides was obtained, when each strain was inoculated together to the fermentation medium from each seed culture in the same inoculum size.

5. The nucleotide productions by No. 21–26–101 or No. 21–26–102 with authentic strains were examined by the mixed culture technique. It was found that production of IMP or guanine nucleotides by Brevibacterium ammoniagenes ATCC 6871 was stimulated remarkably in the presence of No. 21–26–102.     

Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

Hydrogen production of a salt tolerant strain Bacillus sp. B2 from marine intertidal sludge

To isolate a salt tolerant hydrogen-producing bacterium, we used the sludge from the intertidal zone of a bathing beach in Tianjin as inoculum to enrich hydrogen-producing bacteria. The sludge was treated by heat-shock pretreatment with three different temperature (80, 100 and 121°C) respectively. A hydrogen-producing bacterium was isolated from the sludge pretreated at 80°C by sandwich plate technique and identified using microscopic examination and 16S rDNA gene sequence analysis. The isolated bacterium was named as Bacillus sp. B2. The present study examined the hydrogen-producing ability of Bacillus sp. B2. The strain was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum at pH 7.0. The level of hydrogen production was also affected by the salt concentration. Strain B2 has unique capability to adapt high salt concentration. It could produce hydrogen at the salt concentration from 4 to 60‰. The maximum of hydrogen-producing yield of strain B2 was 1.65 ± 0.04 mol H₂/mol glucose (mean ± SE) at an initial pH value of 7.0 in marine culture conditions. Hydrogen production under fresh culture conditions reached a higher level than that in marine ones. As a result, it is likely that Bacillus sp. B2 could be applied to biohydrogen production using both marine and fresh organic waste.     

Diverse Secondary Metabolites Produced by Marine‐Derived Fungus Nigrospora sp. MA75 on Various Culture Media

Bioassay‐guided isolation of a fungal strain Nigrospora sp. MA75, an endophytic fungus obtained from the marine semi‐mangrove plant Pongamia pinnata, which was fermented on three different culture media, resulted in the isolation and identification of seven known compounds, 2, 3 , and 5 – 9 , from a medium containing 3.5% NaCl, while a new compound, 2,3‐didehydro‐19α‐hydroxy‐14‐epicochlioquinone B ( 10 ) was obtained from the medium containing 3.5% NaI. In addition, two new griseofulvin derivatives, 6‐O‐desmethyldechlorogriseofulvin ( 1 ) and 6′‐hydroxygriseofulvin ( 4 ), were isolated and identified from the rice solid medium. Dechlorogriseofulvin ( 2 ) and griseofulvin ( 3 ) were the major components in fermentation extracts of all these culture media, while compounds 1 and 4, 5 and 6 , and 10 were only present in the extract of respective culture medium. The structures of these compounds were elucidated by detailed spectroscopic analysis, and the absolute configuration of 1 was determined by CD measurement. Compounds 9 and 10 exhibited antibacterial activities toward five tested bacterial strains, while compounds 5, 6 , and 8 selectively inhibited MRSA, E. coli, and S. epidermidis, and compound 3 showed moderate activity against V. mali and S. solani. Moreover, compound 10 potently inhibited the growth of MCF‐7, SW1990, and SMMC7721 tumor cell lines with IC₅₀ values of 4, 5, and 7 μg/ml, respectively.     

Degradation of Fumonisin B1 by a Bacterial Strain Isolated from Soil

A mixed microbial culture degrading fumonisin B l was obtained from soil samples using an enrichment culture procedure. A bacterial isolate from the enrichment culture (strain NCB 1492) degraded fumonisin B1 after incubation for 3 h, as indicated by TLC and HPLC analysis. On the basis of the sequence analysis of 16S rDNA, strain NCB 1492 was related to the Delftia/Comamonas group. Thin-layer chromatographic analysis indicated the presence of metabolites in the NCB 1492 culture filtrates after degradation of fumonisin B1 supplied as sole carbon and nitrogen source in phosphate buffer. Four metabolites were identified by mass spectrometry analysis.     

Exploration,antifungal and antiaflatoxigenic activity of halophilic bacteria communities from saline soils of Howze-Soltan playa in Iran

In the present study, halophilic bacteria communities were explored in saline soils of Howze-Soltan playa in Iran with special attention to their biological activity against an aflatoxigenic Aspergillus parasiticus NRRL 2999. Halophilic bacteria were isolated from a total of 20 saline soils using specific culture media and identified by 16S rRNA sequencing in neighbor-joining tree analysis. Antifungal and antiaflatoxigenic activities of the bacteria were screened by a nor-mutant A. parasiticus NRRL 2999 using visual agar plate assay and confirmed by high-performance liquid chromatography. Among a total of 177 halophilic bacteria belonging to 11 genera, 121 isolates (68.3%) inhibited A. parasiticus growth and/or aflatoxin production. The most potent inhibitory bacteria of the genera Bacillus, Paenibacillus and Staphylococcus were distributed in three main phylogenetic clusters as evidenced by 16S rRNA sequence analysis. A. parasiticus growth was inhibited by 0.7–92.7%, while AFB₁ and AFG₁ productions were suppressed by 15.1–98.9 and 57.0–99.6%, respectively. Taken together, halophilic bacteria identified in this study may be considered as potential sources of novel bioactive metabolites as well as promising candidates to develop new biocontrol agents for managing toxigenic fungi growth and subsequent aflatoxin contamination of food and feed in practice.