Tunisian Soil Research Articles

Decolourization of Black Oxidized Olive-Mill Wastewater by a New Tannase-Producing Aspergillus flavus Strain Isolated from Soil

By contaminating a Tunisian soil with black oxidized and sterilized olive-mill wastewaters (OMW), 30 new indigenous fungal soil strains able to overcome the OMW toxicity could be directly selected. Ten of the fungal strains previously isolated were screened for their capability to grow in a liquid culture medium containing oxidized OMW as the only source of carbon and energy. According to these preliminary tests, strain F2 showed the best capability of removing black colour and COD (chemical oxygen demand) and was further identified as Aspergillus flavus. After optimization of batch-liquid culture conditions in the presence of oxidized OMW, the time course of biomass and enzyme production by A. flavus F2 was followed in relation to colour and COD removal. A. flavus F2 could efficiently decolourize and detoxify the black oxidized OMW (58 and 46% of colour and COD removal, respectively, after 6 days of cultivation), concomitantly with the production of tannase (8000 UI/l on day 3).

Efficient Transformation Procedure of a Newly Isolated Streptomyces sp. TN58 Strain Producing Antibacterial Activities

A new aerobic Gram-positive bacterium designated TN58 producing antibacterial activities against Gram-positive and Gram-negative bacteria was isolated from Tunisian soil. The nucleotide sequence of the 16S rRNA gene (1516 bp) of the TN58 strain showed high similarity (96-98%) to the Streptomyces 16S rRNA genes, especially with that of Streptomyces lavendulae which produces the anti-tumor compound mitomycin C, and the cyclic peptide antibiotic, complestatin. Cultural characteristic studies, alignment data of the 16S rRNA gene, and analysis of the nucleotide sequence of a 2.2 kb genomic DNA fragment from TN58 strongly suggested that this strain could be an actinomycete and most probably belongs to the genus Streptomyces. Study of the influence of different nutritional compounds on antibiotic production showed that the highest antibacterial activities were obtained when glycerol at 1% (w/v) was used as sole carbon source in the presence of potassium. In analytical conditions, the application to supernatant culture of the TN58 strain of various extraction and purification steps led to the isolation of two pure active molecules having a retention time of 38.6 and 50.2 min, respectively. TN58 strain was untransformable with the Streptomyces cloning vector pIJ702 via classical polyethylene glycol (PEG) protoplast transformation and previously described Streptomyces electroporation procedures. Transformation was rendered possible by the electroporation technique only after utilization of a preculture medium without sucrose and a regeneration plate containing a low sucrose concentration.

In situ identification and X-ray imaging of microorganisms distribution on the Tatahouine meteorite

Microorganisms were searched for among the complex microstructures observed on the surface of a fragment of the Tatahouine meteorite inherited from the Tunisian soil in which they were buried. In this view, the chemical compositions, particularly the nitrogen, phosphorus, and sulphur compositions, including the sulphur speciation, were investigated using scanning electron microscopy (SEM), electron probe microanalysis (EPMA) mapping, and scanning X-ray microscopy (SXM). A few 2-μm-thick filaments, partly covered by patches of calcite ensuring they were not deposited by a laboratory contamination, were observed by SEM. The EPMA maps show that the portions free of calcite of the filaments have low but constant contents of nitrogen, sulphur, and phosphorus. The SXM maps were recorded at 2473.5, 2478, and 2482.2 eV, which are respectively characteristic for amino acid linked sulphur, sulphite (SO 3 2−), and sulphate (SO 4 2−). The portions of the filaments detected by EPMA are also those that are enriched in amino acid linked sulphur. The calculated (N/S) elemental ratio is consistent with the one of the dehydrated Escherichia coli matter, contrary to the much lower (P/S) elemental ratio. In living cells, the bulk N and S elements are mainly located in large polymers by covalent bonds, whereas a significant amount of P belongs to small and reactive molecules. We thus can propose that the observed microstructures are dehydrated microorganisms, in which most of the elements that were composing the polymers were retained, whereas the small electrolytes and molecules were removed.

Purification and properties of a maltoheptaose- and maltohexaose-forming amylase produced by Bacillus subtilis US116

A new bacterial strain, identified as Bacillus subtilis US116, was isolated from Tunisian soil and selected for its potential production of an atypical amylase with an industrial interest. The identification was founded on physiological tests and molecular techniques related to the 16S rRNA, 23S rRNA genes and intergenic sequences showing the highest similarity of 98% with regions in the complete genome of Bacillus subtilis 168 (accession no. Z99104). This strain produces an atypical amylase that was purified to homogeneity by a combination of acetone precipitation, size exclusion and ion exchange chromatography. The molecular mass of the enzyme is about 60 kDa as determined by SDS–PAGE. Optimal conditions for the activity of the purified enzyme are pH 6 and 65 °C. The half-life duration is about 3 h at 70 °C and 5 h at 65 °C. This enzyme belongs to the endo-type amylases according to the hydrolytic mode study using Ceralpha and Betamyl methods. It is classified as a maltoheptaose- and maltohexaose-forming amylase since it generates about 30% maltohexaose (DP6) and 20% maltoheptaose (DP7) from starch. Moreover, the minimum length of maltosaccharide cleaved by this enzyme was maltoheptaose.

Isolation and purification of an acidic pullulanase type II from newly isolated Bacillus sp. US149

A strain US149 having high pullulanase activity was isolated from Tunisian soil adjacent to hot spring. The cells of this strain were rod shaped, Gram +, non-motile, and could grow only in acidic pH (4.5–5.0) between 37 and 55 °C. The strain is likely to be Bacillus naganoensis since the analysis of the 16S rRNA gene sequence showed highest similarity (96%) with that of B. naganoensis (accession no. AB021193). The enzyme was purified to homogeneity by ion exchange and size exclusion chromatography from cell free culture supernatant. The purified native form had an estimated molecular mass of 200 kDa and it is composed of two subunits each of 95 kDa determined by SDS–PAGE. The purified enzyme had an optimum pH of 5 and retained activity at pH lower than 5. It had half-life duration of 48 and 20 min at 80 and 90 °C, respectively. The enzyme could hydrolyze pullulan, but it was unable to hydrolyze amylose and maltoheptaose. It was demonstrated that the DP3 product of pullulan hydrolysis was isopanose since it was cleaved by α-glucosidase to form glucose and isomaltose. This proved that the PulUS149 is an isopullulanase (pullulan-4-glucanohydrolase, EC 3.2.1.57) classified under pullulanase type II.

Isolation, purification and partial characterization of antibacterial activities produced by a newly isolated Streptomyces sp. US24 strain

A new actinomycete strain designated US24 producing antibacterial activities against Gram-positive and Gram-negative bacteria was isolated from Tunisian soil. Culture characteristic studies strongly suggested that the US24 strain belonged to the genus Streptomyces. Analysis of the nucleotide sequence of the 16S rRNA gene of the Streptomyces sp. US24 strain showed high similarity (98%) with the 16S rRNA gene of Streptomyces caelestis which produces two antibiotics, niddamycin and celesticetin. Study of the influence of different nutritional compounds on antibiotic biosynthesis showed that the highest antibacterial activities were obtained when starch at 1% (w/v) was used as sole carbon source in the presence of traces of mineral oligoelements. Application to the supernatant culture of the Streptomyces sp. US24 strain of various separation steps led to isolation of two pure active molecules having a retention time of 34 and 37.26 min, respectively. P 34 min possessed antibacterial activity against Gram-positive and Gram-negative bacteria, whereas P 37.26 min inhibited only Gram-positive bacteria. Partial characterization of the P 34 min molecule using spectroscopic studies showed that this active molecule is different from the two antibiotics produced by the S. caelestis strain.

Genotypic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of Phaseolus vulgaris L. grown in Tunisian soils

One hundred and sixty isolates of rhizobia were sampled from the root nodules of common bean (Phaseolus vulgaris L.) cultivated in Tunisian soil samples originating from three geographically distinct fields. Plasmid profiling was used as a primary method to rapidly screen the isolates, and then 38 plasmid types were recorded among the 160 isolates. A sample representing the majority of plasmid types was chosen for further characterization by restriction fragment length polymorphism (RFLP) analysis of genomic DNA using chromosomal and symbiotic gene probes, and by their ability to nodulate a potential alternative host, Leucaena leucocephala. One third of the isolates showed a high similarity to Rhizobium gallicum isolated from common bean in France, another third showed the same characteristics as the R. etli-R. leguminosarum group, while the remaining isolates could not be related to any of the five species nodulating bean. When reexamined for nodulation, some of these isolates, showing similarities to R. tropici and Agrobacterium with respect to colony morphology and growth in different media, failed to nodulate their original host. The R. gallicum-like isolates, R. etli-like isolates, and R. leguminosarum-like isolates were recovered from regions where bean is frequently grown, while in fields which had not been cultivated with beans for at least the 10 previous years, solely unrecognized taxa of ineffective isolates were recovered. We detected variations in the symbiotic regions, but certain pSym RFLP patterns for nifH were conserved between Tunisian, French, and Austrian populations of bean rhizobia. Evaluation of symbiotic effectiveness showed that R. gallicum-like isolates and R. etli-like isolates were effective, whereas some R. leguminosarum-like isolates were ineffective. Furthermore, effective isolates were also found among the unrecognized taxa.

Characterization and molecular cloning of thermostable alpha-amylase from Streptomyces sp.To1

A new thermophilic Streptomyces sp. TO1, isolated from Tunisian soil, produced a thermostable alpha-amylase and pullulanase. The gene encoding for the alpha-amylase activity was cloned into the multicopy cloning plasmid pLM1 using S. lividans ZX1 as host strain. The ZX1 / pLM1 strain has the same activity than the initial TO1 strain and about 25 fold higher activity than the ZX1 strain. This alpha-amylase has an optimum of pH and temperature at 6 and 70 °C respectively.

96/01782 Anaerobic digestion of municipal solid waste: A modern waste disposal option on the verge of breakthrough

It is obvious that the application of solid waste compost improves the soil fertility. These wastes, however, may also have some negative effects on the agricultural environment due to their metal content. This research aimed at evaluating the influence of Tunisian municipal solid waste compost and farmyard manure on some chemical properties and the distribution of heavy metals in a calcareous Tunisian soil (clayey–loamy soil). A field plot experiment, without vegetation, was installed since 1999 at the experimental farm of the Agronomic National Institute of Tunis (INAT) in the region of Mornag (20 km south of Tunis, Tunisia). During 5 years, the field received yearly the following treatments: 0, 40, 80 and 120 t/ha of municipal solid waste compost and 0, 40 and 120 t/ha of manure. The fractionation of heavy metals in the soil was evaluated after 5 years using a sequential extraction procedure. The application of the two amendments was found to increase the content of organic matter, the total nitrogen content and the electrical conductivity, whereas it slightly decreased the soil pH. The addition of manure did not have a significant effect on the accumulation of heavy metals in the soil, whereas compost application increased the total concentration of heavy metals in the soil. The distribution of heavy metals between the different fractions in untreated and treated soils showed the residual fraction to be dominant, followed by the fraction bound to Fe and Mn oxides. The amount of Cu bound to the organic fraction increased with the application rate, which is probably caused by the formation of organic complexes. For the other metals, the increase of the association with organic matter is very limited. The application of compost moreover increases the amount of Zn associated with Fe and Mn oxides. The “Mobility Factor (MF)” was quite low and did not change after the 5-year application of the two organic amendments. It always remained lower than 10%, although for Cd it amounted to 17%.

Characterization of Goethite and Hematite in a Tunisian Soil Profile By Mössbauer Spectroscopy

AbstractAs part of the characterization of a Tunisian red soil profile, six samples, taken at different depths, were investigated by Mössbauer spectroscopy at room temperature and at 80 K to obtain information about the various types of Fe oxides present. By considering magnetic hyperfine field distributions, the spectra of goethite and hematite were well resolved. Chemical analyses of the samples revealed a partial substitution of Fe by Al and Mn. The spectral behavior of the goethite was predominantly influenced by crystallinity and amount of Al substitution which resulted in a reduction of the magnetic hyperfine field. The effect of Mn substitution was much more pronounced in the hematite spectrum as a consequence of a stronger suppression of the Morin transition by Mn than by Al.