Bioremediation of soils saturated with spilled crude oil.

A desert soil sample was saturated with crude oil (17.3%, w/w) and aliquots were diluted to different extents with either pristine desert or garden soils. Heaps of all samples were exposed to outdoor conditions through six months, and were repeatedly irrigated with water and mixed thoroughly. Quantitative determination of the residual oil in the samples revealed that oil-bioremediation in the undiluted heaps was nearly as equally effective as in the diluted ones. One month after starting the experiment. 53 to 63% of oil was removed. During the subsequent five months, 14 to 24% of the oil continued to be consumed. The dynamics of the hydrocarbonoclastic bacterial communities in the heaps was monitored. The highest numbers of those organisms coordinated chronologically with the maximum oil-removal. Out of the identified bacterial species, those affiliated with the genera Nocardioides (especially N. deserti), Dietzia (especially D. papillomatosis), Microbacterium, Micrococcus, Arthrobacter, Pseudomonas, Cellulomonas, Gordonia and others were main contributors to the oil-consumption. Some species, e.g. D. papillomatosis were minor community constituents at time zero but they prevailed at later phases. Most isolates tolerated up to 20% oil, and D. papillomatosis showed the maximum tolerance compared with all the other studied isolates. It was concluded that even in oil-saturated soil, self-cleaning proceeds at a normal rate. When pristine soil receives spilled oil, indigenous microorganisms suitable for dealing with the prevailing oil-concentrations become enriched and involved in oil-biodegradation.

Effect of halotolerant rhizobacteria isolated from halophytes on the growth of sugar beet (Beta vulgaris L.) under salt stress.

Utilization of rhizobacteria that have associated with plant roots in harsh environments could be a feasible strategy to deal with limits to agricultural production caused by soil salinity. Halophytes occur naturally in high-salt environments, and their roots may be associated with promising microbial candidates for promoting growth and salt tolerance in crops. This study aimed to isolate efficient halotolerant plant-growth-promoting rhizobacterial strains from halophytes and evaluate their activity and effects on sugar beet (Beta vulgaris L.) growth under salinity stress. A total of 23 isolates were initially screened for their ability to secrete 1-aminocyclopropane-1-carboxylate deaminase (ACD) as well as other plant-growth-promoting characteristics and subsequently identified by sequencing the 16S rRNA gene. Three isolates, identified as Micrococcus yunnanensis, Planococcus rifietoensis and Variovorax paradoxus, enhanced salt stress tolerance remarkably in sugar beet, resulting in greater seed germination and plant biomass, higher photosynthetic capacity and lower stress-induced ethylene production at different NaCl concentrations (50-125 mM). These results demonstrate that salinity-adapted, ACD-producing bacteria isolated from halophytes could promote sugar beet growth under saline stress conditions.

[Secondary metabolites from a deep-sea-derived actinomycete Micrococcus sp. R21].

To investigate cytotoxic secondary metabolites of Micrococcus sp. R21, an actinomycete isolated from a deep-sea sediment (-6 310 m; 142 degrees 19. 9' E, 10 degrees 54. 6' N) of the Western Pacific Ocean, column chromatography was introduced over silica gel, ODS, and Sephadex LH-20. As a result, eight compounds were obtained. By mainly detailed analysis of the NMR data, their structures were elucidated as cyclo(4-hydroxy-L-Pro-L-leu) (1), cyclo(L-Pro-L-Gly) (2), cyclo( L-Pro-L-Ala) (3), cyclo( D-Pro-L-Leu) (4), N-ß-acetyltryptamine (5), 2-hydroxybenzoic acid (6), and phenylacetic acid (7). Compound 1 exhibited weak cytotoxic activity against RAW264. 7 cells with IC50 value of 9.1 µmol x L(-1).

Bioremediation potential of microorganisms derived from petroleum reservoirs.

Bacterial strains and metagenomic clones, both obtained from petroleum reservoirs, were evaluated for petroleum degradation abilities either individually or in pools using seawater microcosms for 21 days. Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) analyses were carried out to evaluate crude oil degradation. The results showed that metagenomic clones 1A and 2B were able to biodegrade n-alkanes (C14 to C33) and isoprenoids (phytane and pristane), with rates ranging from 31% to 47%, respectively. The bacteria Dietzia maris CBMAI 705 and Micrococcus sp. CBMAI 636 showed higher rates reaching 99% after 21 days. The metagenomic clone pool biodegraded these compounds at rates ranging from 11% to 45%. Regarding aromatic compound biodegradation, metagenomic clones 2B and 10A were able to biodegrade up to 94% of phenanthrene and methylphenanthrenes (3-MP, 2-MP, 9-MP and 1-MP) with rates ranging from 55% to 70% after 21 days, while the bacteria Dietzia maris CBMAI 705 and Micrococcus sp. CBMAI 636 were able to biodegrade 63% and up to 99% of phenanthrene, respectively, and methylphenanthrenes (3-MP, 2-MP, 9-MP and 1-MP) with rates ranging from 23% to 99% after 21 days. In this work, isolated strains as well as metagenomic clones were capable of degrading several petroleum compounds, revealing an innovative strategy and a great potential for further biotechnological and bioremediation applications.

The mode of action of the lantibiotic lacticin 3147--a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II.

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.

Production, purification, and characterization of micrococcin GO5, a bacteriocin produced by Micrococcus sp. GO5 isolated from kimchi.

Strain GO5, a bacteriocin-producing bacterium, was isolated from green onion kimchi and identified as Micrococcus sp. The bacteriocin, micrococcin GO5, displayed a broad spectrum of inhibitory activity against a variety of pathogenic and nonpathogenic microorganisms, as tested by the spot-on-lawn method; its activity spectrum was almost identical to that of nisin. Micrococcin GO5 was inactivated by trypsin (whereas nisin was not) and was completely stable at 100 degrees C for 30 min and in the pH range of 2.0 to 7.0. Micrococcin GO5 exhibited a typical mode of bactericidal activity against Micrococcus flavus ATCC 10240. It was purified to homogeneity through ammonium sulfate precipitation, ultrafiltration, and CM-Sepharose column chromatography. The molecular mass of micrococcin GO5 was estimated to be about 5.0 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ activity assay with the indicator organism. The amino acid sequence of micrococcin GO5 lacks lanthionine and beta-methyllanthionine and is rich in hydrophobic amino acids and glycine, providing the basis for the high heat stability of this bacteriocin. The N-terminal amino acid sequence of micrococcin GO5 is Lys-Lys-Ser-Phe-Cys-Gln-Lys, and no homology to bacteriocins reported previously was observed in the amino acid composition or N-terminal amino acid sequence. Based on the physicochemical properties, small molecular size, and inhibition of Listeria monocytogenes, micrococcin GO5 has been placed with the class II bacteriocins, but its broad spectrum of activity differs from that of other bacteriocins in this class.

Studies on the effect of inoculation of activated sludge with bacteria actively degrading hydrocarbons on the biodegradation of petroleum products.

Eighteen strains of bacteria were isolated from activated sludge purifying petroleum-refining wastewaters. These strains were plated on solidified mineral medium supplemented with oil fraction in concentration 1000 mg/l. Four of the strains that grew best in the presence of oil were selected for further studies. The strains were identified based on Bonde's scheme and microscopic observations. Three of them belonged to the genus Arthrobacter and one to the genus Micrococcus. Stationary cultures of single strains and their mixtures were set up in mineral medium containing oil (sterile and non-sterile) as sole carbon source in concentration 1000 mg/l. The oils were found to be removed the most efficiently by a mixture of the strains. After 14 days of culture the amount of oil was utilized by from 63 to 95%. In the next stage of the studies the bacteria were used to inoculate activated sludge. Stationary cultures of the activated sludge were set up in mineral medium with oil. The utilisation of petroleum products by non-inoculated activated sludge (control), activated sludge inoculated with a single strain or a mixture of all four strains was examined. In both inoculated activated sludge cultures approximately 80% of the oils were removed, compared to 60% in the control activated sludge. Therefore, inoculated activated sludge showed 20% higher effectiveness of removal of petroleum derivatives.

Lysine production from hydrocarbon by Micrococcus varians 2Fa.

A bacterium isolated from Assam (India) soil was found to accumulale l-lysine from hydrocarbon and was identified as a strain of Micrococcus varians. The strain is able to grow and accumulate lysine in a purely synthetic medium though supplementation of the synthetic medium with casamino acids significantly improves the yield. The yield of l-lysine under optimal conditions was found to be 2.6 g X 1(-1) of the compound isolated in crystalline form.

Valine production from hydrocarbon by Micrococcus varians.

A bacterium isolated from Assam (India) soil was found to accumulate L-valine in the growth medium and was identified as Micrococcus varians. The strain grew and accumulated valine in a purely synthetic medium, but supplementation with either casamino acids or yeast extract or with both, improved the yield. The entire fermentation period could be divided into a growth phase and a production (phase which could be prolonged by adjustment of pH to neutral range. Among the different hydrocarbon and nitrogen sources tested straight run gas-oil and ammonium sulphate, respectively, were found most suitable. Antibiotics inhibited growth but stimulated extracellular valine accumulation. Vitamins stimulated growth and valine yield and an inoculum level of 10% was found to be optimal. The yield of L-valine under optimal conditions was 2.95 g/L.

Cholesterol incorporation into bacterial membranes.

The wall-covered bacteria Micrococcus lysodeikticus, Bacillus megaterium, and Proteus mirabilis incorporated exogenous cholesterol into their cytoplasmic membrane in quantities resembling those incorporated by sterol-nonrequiring mycoplasmas. Cholesterol incorporation into the outer membrane of P. mirabilis was much more restricted than into the cytoplasmic membrane.

[Effect of the stereoisomers of glutamic acid on the vital activity of auxotrophic mutant producers of lysine]. / Vliianie stereoizomerov glutaminovoi kisloty na zhiznedeiatel'-nost' auksotrofnykh mutantov--produtsentov lizina

Studies of L, D and DL-forms of glutamic acid as the sole nitrogen source or as a supplement to the major nitrogen source--ammonium sulphate have demonstrated that in the first case all forms of glutamic acid are assimilated by auxotrophic mutants--lysine producers. However, the lysine synthesis was very low, when L-glutamic acid was used as the only nitrogen source. Glutamic acid at a concentration of 0.01 M applied as a supplement to the major source of nitrogen acted as a stimulator of lysine synthesis, slightly inhibiting biomass increment. That was true of every form of glutamic acid, especially of DL-glutamic acid.

Microbial utilization of crude oil.

The utilization of two crude oil samples of different quality at 4 and 30 C has been studied by using pure and mixed bacterial cultures obtained by enrichment procedures. Growth, emulsification, and utilization occurred readily at both temperatures. The crude oil residue is increased in specific gravity and readily sediments out of solution. A comparison of the chemical analysis of the oils by liquid and gas-liquid chromatographic procedures before and after growth showed that the n-saturate fraction had been preferentially used. Some utilization of the aromatic fraction also occurred. Enrichments obtained with a high-quality crude oil were not as effective in utilizing a lower quality crude oil as sole carbon source as a population enriched on the low-quality crude oil.

Characterization of the late steps of microbial heme synthesis: conversion of coproporphyrinogen to protoporphyrin.

Cell-free extracts of various cytochrome-containing, heterotrophic microorganisms were examined for ability to convert coproporphyrinogen to protoporphyrin. Extracts of Escherichia coli and Pseudomonas denitrificans readily accumulated large amounts of protoporphyrin when assayed under aerobic conditions. However, protoporphyrin did not accumulate under either aerobic or anaerobic conditions of assay or in the presence of various supplements in extracts of the aerobe Micrococcus lysodeikticus, the facultative anaerobe Staphylococcus aureus, or the anaerobe Vibrio succinogenes. Protoporphyrin also accumulated when extracts of E. coli and P. denitrificans were incubated aerobically with the early heme precursor, delta-amino levulinic acid (ALA). This protoporphyrin accumulation was markedly stimulated by the iron chelator, o-phenanthroline. Extracts of S. aureus and M. lysodeikticus accumulated coproporphyrin, but not protoporphyrin when incubated with ALA. The enzyme system in extracts of E. coli which converts coproporphyrinogen to protoporphyrin under aerobic conditions of assay was also partially characterized. This conversion was stimulated by the iron chelator, o-phenanthroline, the respiratory inhibitor, cyanide, and the reducing agent, thioglycolate. Dialysis of the extract did not diminish enzyme activity. Certain alternate electron acceptors and nitrite caused a marked inhibition of the conversion. These results indicate that this late step in heme synthesis, the conversion of coproporphyrinogen to protoporphyrin, can be readily demonstrated in extracts of some, but not all, cytochrome-containing bacteria and that the aerobic conversion in E. coli exhibits many characteristics similar to those demonstrated for the aerobic conversion previously studied in liver mitochondria.