Extremophilic Bacterium Halomonas desertis G11 as a Cell Factory for Poly-3-Hydroxybutyrate-co-3-Hydroxyvalerate Copolymer's Production.

Microbial polyhydroxyalkanoates (PHA) are biodegradable and biocompatible bio-based polyesters, which are used in various applications including packaging, medical and coating materials. In this study, an extremophilic hydrocarbonoclastic bacterium, previously isolated from saline sediment in the Tunisian desert, has been investigated for PHA production. The accumulation of intracellular PHA granules in Halomonas desertis G11 was detected by Nile blue A staining of the colonies. To achieve maximum PHA yield by the strain G11, the culture conditions were optimized through response surface methodology (RSM) employing a Box-Behnken Design (BBD) with three independent variables, namely, substrate concentration (1-5%), inoculum size (1-5%) and incubation time (5-15 days). Under optimized conditions, G11 strain produced 1.5 g/L (68% of DCW) of PHA using glycerol as a substrate. Application of NMR (1H and 13C) and FTIR spectroscopies showed that H. desertis accumulated PHA is a poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). The genome analysis revealed the presence of typical structural genes involved in PHBV metabolism including phaA, phaB, phaC, phaP, phaZ, and phaR, coding for acetyl-CoA acetyltransferase, acetoacetyl-CoA reductase, class I polyhydroxyalkanoates synthases, phasin, polyhydroxyalkanoates depolymerase and polyhydroxyalkanoates synthesis repressor, respectively. Glycerol can be metabolized to 1) acetyl-CoA through the glycolysis pathway and subsequently converted to the 3HB monomer, and 2) to propionyl-CoA via the threonine biosynthetic pathway and subsequently converted to the 3HV monomer. In silico analysis of PhaC1 from H. desertis G11 indicated that this enzyme belongs to Class I PHA synthase family with a "lipase box"-like sequence (SYCVG). All these characteristics make the extremophilic bacterium H. desertis G11 a promising cell factory for the conversion of bio-renewable glycerol to high-value PHBV.

Genome analysis provides insights into crude oil degradation and biosurfactant production by extremely halotolerant Halomonas desertis G11 isolated from Chott El-Djerid salt-lake in Tunisian desert.

Here, we report the genomic features and the bioremediation potential of Halomonas desertis G11, a new halophilic species which uses crude oil as a carbon and energy source and displays intrinsic resistance to salt stress conditions (optimum growth at 10% NaCl). G11 genome (3.96 Mb) had a mean GC content of 57.82%, 3622 coding sequences, 480 subsystems and 64 RNA genes. Annotation predicted 38 genes involved in osmotic stress including the biosynthesis of osmoprotectants glycine-betaine, ectoine and osmoregulated periplasmic glucans. Genome analysis revealed also the versatility of the strain for emulsifying crude oil and metabolizing hydrocarbons. The ability of G11 to degrade crude oil components and to secrete a glycolipid biosurfactant with satisfying properties was experimentally confirmed and validated. Our results help to explain the exceptional capacity of G11 to survive at extreme desertic conditions, and highlight the metabolic features of this organism that has biotechnological and ecological potentialities.

Cultivation-dependent assessment, diversity, and ecology of haloalkaliphilic bacteria in arid saline systems of southern Tunisia.

Haloalkaliphiles are polyextremophiles adapted to grow at high salt concentrations and alkaline pH values. In this work, we isolated 122 haloalkaliphilic bacteria upon enrichments of 23 samples from 5 distinct saline systems of southern Tunisia, growing optimally in media with 10% salt and at pH 10. The collection was classified into 44 groups based on the amplification of the 16S-23S rRNA internal transcribed spacers (ITS-PCR). Phylogenetic analysis and sequencing of the 16S rRNA genes allowed the identification of 13 genera and 20 distinct species. Three gram-positive isolates showing between 95 and 96% of 16S rRNA sequence homology with Bacillus saliphilus could represent new species or genus. Beside the difference in bacterial diversity between the studied sites, several species ecological niches correlations were demonstrated such as Oceanobacillus in salt crust, Nesterenkonia in sand, and Salinicoccus in the rhizosphere of the desert plant Salicornia. The collection was further evaluated for the production of extracellular enzymes. Activity tests showed that gram-positive bacteria were mostly active, particularly for protease, lipase, DNase, and amylase production. Our overall results demonstrate the huge phenotypic and phylogenetic diversity of haloalkaliphiles in saline systems of southern Tunisia which represent a valuable source of new lineages and metabolites.

Uneven distribution of Halobacillus trueperi species in arid natural saline systems of Southern Tunisian Sahara.

The genetic diversity of a collection of 336 spore-forming isolates recovered from five salt-saturated brines and soils (Chott and Sebkhas) mainly located in the hyper-arid regions of the southern Tunisian Sahara has been assessed. Requirements and abilities for growth at a wide range of salinities\ showed that 44.3 % of the isolates were extremely halotolerant, 23 % were moderate halotolerant, and 32.7 % were strict halophiles, indicating that they are adapted to thrive in these saline ecosystems. A wide genetic diversity was documented based on 16S-23S rRNA internal transcribed spacer fingerprinting profiles (ITS) and 16S rRNA gene sequences that clustered the strains into seven genera: Bacillus, Gracilibacillus, Halobacillus, Oceanobacillus, Paenibacillus, Pontibacillus, and Virgibacillus. Halobacillus trueperi was the most encountered species in all the sites and presented a large intraspecific diversity with a multiplicity of ITS types. The most frequent ITS type included 42 isolates that were chosen for assessing of the intraspecific diversity by BOX-PCR fingerprinting. A high intraspecific microdiversity was documented by 14 BOX-PCR genotypes whose distribution correlated with the strain geographic origin. Interestingly, H. trueperi isolates presented an uneven geographic distribution among sites with the highest frequency of isolation from the coastal sites, suggesting a marine rather than terrestrial origin of the strains. The high frequency and diversity of H. trueperi suggest that it is a major ecosystem-adapted microbial component of the Tunisian Sahara harsh saline systems of marine origin.