Mode of uptake of insoluble solid substrates by microorganisms. II: Uptake of solid n-alkanes by yeast and bacterial species

Using two species of yeast and one of bacterium, evidence has ben obtained which indicates that the microbial uptake of solid alkane powders occurs primarily through a substrate solubilization mechanism. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited the growth of these organisms on alkane powder; the inhibition could be removed vai a supply of artificially solubilized alkane. One of the yeast strians, which was a mutant incapable of growing on solid alkane powder and liquid alkane, could grow very well on artifically solubilized alkanes. It was demonstrated that the solid alkane solubilization rate during microbial growth could satisfactorily account for the maximal alkane uptake rate actully observed during growth. The specificity of solubilization for the solid alkane used as the growth substrate was demonstrated.     

Uptake of volatilen-alkanes byPseudomonas PG-I

Using a bacterial speciesPseudomonas PG-1, evidence has been obtained which indicates that uptake ofn-pentane ton-octane by microbial cells takes place primarily from the gas phase either directly orvia the aqueous phase. Specific growth rate increased along with the increase in substrate concentration but above the alkane concentration of 0.3% by volume, specific growth rate decreased indicating substrate inhibition of growth. In the case of less volatile alkanes,n-nonane andn-decane, substrate transfer is predominantly through substrate solubilization system elaborated by the cells. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited growth on these two alkanes but had negligible effect on growth onn-pentane ton-octane.     

Rapid identification of biosurfactant-producing bacterial strains using a cell surface hydrophobicity technique

Rapid identification of biosurfactant-producing bacterial strains was achieved by assaying cell surface hydrophobicity which had a direct correlation with biosurfactant production by Serratia marcescens, Pseudomonas aeruginosa, Bacillus pumilus, B. laterosporus, Acineto- bacter calcoaceticus, Escherichia coli and Staphylococcus aureus. These properties namely, Hydrophobic Interaction Chromatography, Salt Aggregation Test, Bacterial Adherence To Hydrocarbon and adhesion to polystyrene by Replica Plate test, provide a simple means for identifying bacteria associated with the production of biosurfactants.     

Biosurfactants in agriculture

Agricultural productivity to meet growing demands of human population is a matter of great concern for all countries. Use of green compounds to achieve the sustainable agriculture is the present necessity. This review highlights the enormous use of harsh surfactants in agricultural soil and agrochemical industries. Biosurfactants which are reported to be produced by bacteria, yeasts, and fungi can serve as green surfactants. Biosurfactants are considered to be less toxic and eco-friendly and thus several types of biosurfactants have the potential to be commercially produced for extensive applications in pharmaceutical, cosmetics, and food industries. The biosurfactants synthesized by environmental isolates also has promising role in the agricultural industry. Many rhizosphere and plant associated microbes produce biosurfactant; these biomolecules play vital role in motility, signaling, and biofilm formation, indicating that biosurfactant governs plant–microbe interaction. In agriculture, biosurfactants can be used for plant pathogen elimination and for increasing the bioavailability of nutrient for beneficial plant associated microbes. Biosurfactants can widely be applied for improving the agricultural soil quality by soil remediation. These biomolecules can replace the harsh surfactant presently being used in million dollar pesticide industries. Thus, exploring biosurfactants from environmental isolates for investigating their potential role in plant growth promotion and other related agricultural applications warrants details research. Conventional methods are followed for screening the microbial population for production of biosurfactant. However, molecular methods are fewer in reaching biosurfactants from diverse microbial population and there is need to explore novel biosurfactant from uncultured microbes in soil biosphere by using advanced methodologies like functional metagenomics.     

Metabolism of 2-Chloro-4-Nitroaniline via Novel Aerobic Degradation Pathway by Rhodococcus sp. Strain MB-P1

2-chloro-4-nitroaniline (2-C-4-NA) is used as an intermediate in the manufacture of dyes, pharmaceuticals, corrosion inhibitor and also used in the synthesis of niclosamide, a molluscicide. It is marked as a black-listed substance due to its poor biodegradability. We report biodegradation of 2-C-4-NA and its pathway characterization by Rhodococcus sp. strain MB-P1 under aerobic conditions. The strain MB-P1 utilizes 2-C-4-NA as the sole carbon, nitrogen, and energy source. In the growth medium, the degradation of 2-C-4-NA occurs with the release of nitrite ions, chloride ions, and ammonia. During the resting cell studies, the 2-C-4-NA-induced cells of strain MB-P1 transformed 2-C-4-NA stoichiometrically to 4-amino-3-chlorophenol (4-A-3-CP), which subsequently gets transformed to 6-chlorohydroxyquinol (6-CHQ) metabolite. Enzyme assays by cell-free lysates prepared from 2-C-4-NA-induced MB-P1 cells, demonstrated that the first enzyme in the 2-C-4-NA degradation pathway is a flavin-dependent monooxygenase that catalyzes the stoichiometric removal of nitro group and production of 4-A-3-CP. Oxygen uptake studies on 4-A-3-CP and related anilines by 2-C-4-NA-induced MB-P1 cells demonstrated the involvement of aniline dioxygenase in the second step of 2-C-4-NA degradation. This is the first report showing 2-C-4-NA degradation and elucidation of corresponding metabolic pathway by an aerobic bacterium.     

Matrix-assisted laser desorption ionization--time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge

An innovative method was developed for rapid sensitive detection and efficient structural characterization of lipopeptide biosurfactants by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cells and crude culture filtrates as targets in combination with surface tension measurements. This was done for a bacterial strain that was isolated from petroleum sludge and efficiently produces biosurfactants. This organism was identified by using biochemical, physiological, and genetic parameters as a Bacillus subtilis strain, designated B. subtilis C-1. This assignment was supported by a mass spectrometric investigation of the secondary metabolite spectrum determined by whole-cell MALDI-TOF mass spectrometry, which revealed three lipopeptide complexes, the surfactins, the iturins, and the fengycins, which are well-known biosurfactants produced by B. subtilis strains. These compounds were structurally characterized by in situ structure analysis by using postsource decay MALDI-TOF mass spectrometry. The isoforms were separated by miniaturized high-resolution reversed-phase high-performance liquid chromatography for mass spectrometric characterization. Iturin compounds which contain unusual fatty acid components were detected.     

Potential commercial applications of microbial surfactants

Surfactants are surface-active compounds capable of reducing surface and interfacial tension at the interfaces between liquids, solids and gases, thereby allowing them to mix or disperse readily as emulsions in water or other liquids. The enormous market demand for surfactants is currently met by numerous synthetic, mainly petroleum-based, chemical surfactants. These compounds are usually toxic to the environment and non-biodegradable. They may bio-accumulate and their production, processes and by-products can be environmentally hazardous. Tightening environmental regulations and increasing awareness for the need to protect the ecosystem have effectively resulted in an increasing interest in biosurfactants as possible alternatives to chemical surfactants. Biosurfactants are amphiphilic compounds of microbial origin with considerable potential in commercial applications within various industries. They have advantages over their chemical counterparts in biodegradability and effectiveness at extreme temperature or pH and in having lower toxicity. Biosurfactants are beginning to acquire a status as potential performance-effective molecules in various fields. At present biosurfactants are mainly used in studies on enhanced oil recovery and hydrocarbon bioremediation. The solubilization and emulsification of toxic chemicals by biosurfactants have also been reported. Biosurfactants also have potential applications in agriculture, cosmetics, pharmaceuticals, detergents, personal care products, food processing, textile manufacturing, laundry supplies, metal treatment and processing, pulp and paper processing and paint industries. Their uses and potential commercial applications in these fields are reviewed. Received: 29 July 1999 / Received revision: 8 November 1999 / Accepted: 9 November 1999     

Aerobic Degradation of N-Methyl-4-Nitroaniline (MNA) by Pseudomonas sp. Strain FK357 Isolated from Soil

N-Methyl-4-nitroaniline (MNA) is used as an additive to lower the melting temperature of energetic materials in the synthesis of insensitive explosives. Although the biotransformation of MNA under anaerobic condition has been reported, its aerobic microbial degradation has not been documented yet. A soil microcosms study showed the efficient aerobic degradation of MNA by the inhabitant soil microorganisms. An aerobic bacterium, Pseudomonas sp. strain FK357, able to utilize MNA as the sole carbon, nitrogen, and energy source, was isolated from soil microcosms. HPLC and GC-MS analysis of the samples obtained from growth and resting cell studies showed the formation of 4-nitroaniline (4-NA), 4-aminophenol (4-AP), and 1, 2, 4-benzenetriol (BT) as major metabolic intermediates in the MNA degradation pathway. Enzymatic assay carried out on cell-free lysates of MNA grown cells confirmed N-demethylation reaction is the first step of MNA degradation with the formation of 4-NA and formaldehyde products. Flavin-dependent transformation of 4-NA to 4-AP in cell extracts demonstrated that the second step of MNA degradation is a monooxygenation. Furthermore, conversion of 4-AP to BT by MNA grown cells indicates the involvement of oxidative deamination (release of NH₂ substituent) reaction in third step of MNA degradation. Subsequent degradation of BT occurs by the action of benzenetriol 1, 2-dioxygenase as reported for the degradation of 4-nitrophenol. This is the first report on aerobic degradation of MNA by a single bacterium along with elucidation of metabolic pathway.