Optimization of thermostable proteases production under agro-wastes solid-state fermentation by a new thermophilic Mycothermus thermophilus isolated from a hydrothermal spring Hammam Debagh, Algeria.

The present work investigates for the first time the presence and isolation of the thermophilic fungi from hydrothermal spring situated at the locality of Guelma, in the Northeast of Algeria. The production of the thermostable proteases and the optimization of culture conditions under agro-wastes solid-state fermentation to achieve optimal production capacity were explored. A statistical experimental approach consisting of two designs was used to determine the optimum culture conditions and to attain the greatest enzyme production. Besides, different agricultural wastes were initially evaluated as a substrate, whereby wheat bran was selected for enzyme production by the isolate under solid-state conditions. The isolate thermophilic fungi were identified as Mycothermus thermophilus by sequencing the ITS region of the rDNA (NCBI Accession No: MK770356.1). Among the various screened variables: the temperature, the inoculum size, and the moisture were proved to have the most significant effects on protease activity. Employing two-level fractional Plackett-Burman and a Box-Behnken designs statistical approach helped in identifying optimum values of screened factors and their interactions. The analysis showed up 6.17-fold improvement in the production of proteases (~1187.03 U/mL) was achieved under the optimal conditions of moisture content 47%, inoculum 5 × 105 spores/g, and temperature at 42 °C. These significant findings highlight the importance of the statistical design in isolation of Mycothermus thermophilus species from a specific location as well as identifying the optimal culture conditions for maximum yield.

In vitro and in vivo characterization of plant growth promoting Bacillus strains isolated from extreme environments of Eastern Algeria.

This report is to our knowledge the first to study plant growth promotion and biocontrol characteristics of Bacillus isolates from extreme environments of Eastern Algeria. Seven isolates of 14 (50 %) were screened for their ability to inhibit growth of some phytopathogenic fungi on PDA and some roots exudates. The bacteria identification based on 16S r-RNA and gyrase-A gene sequence analysis showed that 71 % of the screened isolates belonged to Bacillus amyloliquefaciens and the rest were closely related to B. atrophaeus and B. mojavensis. Most of them had high spore yields (22 × 10(8)-27 × 10(8) spores/ml). They produced protease and cellulase cell wall-degrading enzymes while the chitinase activity was only observed in the B. atrophaeus (6SEL). A wide variety of lipopeptides homologous was detected by liquid chromatography-electrospray ionization-mass spectrometry analysis. Interestingly, some additional peaks with new masses were characterized, which may correspond to new fengycin classes. The isolates produced siderophores and indole-3- acetic acid phytohormone. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. atrophaeus (6SEL) significantly increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05). These results suggest that these isolates may be used further as bio-inoculants to improve crop systems.

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere.

The phenotypic and genotypic diversity of the plant growth promoting Bacillus genus have been widely investigated in the rhizosphere of various agricultural crops. However, to our knowledge this is the first report on the Bacillus species isolated from the rhizosphere of Calendula officinalis. 15 % of the isolated bacteria were screened for their important antifungal activity against Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cladosporium cucumerinium and Alternaria alternata. The bacteria identification based on 16S r-RNA and gyrase-A genes analysis, revealed strains closely related to Bacillus amyloliquefaciens, B. velezensis, B. subtilis sub sp spizezenii and Paenibacillus polymyxa species. The electro-spray mass spectrometry coupled to liquid chromatography (ESI-LC MS) analysis showed that most of the Bacillus isolates produced the three lipopeptides families. However, the P. polymyxa (18SRTS) didn't produce any type of lipopeptides. All the tested Bacillus isolates produced cellulase but the protease activity was observed only in the B. amyloliquefaciens species (9SRTS). The Salkowsky colorimetric test showed that the screened bacteria synthesized 6-52 µg/ml of indole 3 acetic acid. These bacteria produced siderophores with more than 10 mm wide orange zones on chromazurol S. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. amyloliquefaciens (9SRTS) had no significant (P > 0.05) effect on the pre-germination of the chickpea seeds. However, it increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05).These results suggested that the Bacillus strains isolated in this work may be further used as bioinoculants to improve the production of C. officinalis and other crop systems.