Hossein Shahbani Zahiri

Biosurfactant-producing bacterium, Pseudomonas aeruginosa MA01 isolated from spoiled apples: Physicochemical and structural characteristics of isolated biosurfactant

An extensive investigation was conducted to isolate indigenous bacterial strains with outstanding performance for biosurfactant production from different types of spoiled fruits, food-related products and food processing industries. An isolate was selected from 800 by the highest biosurfactant yield in soybean oil medium and it was identified by 16S rRNA and the two most relevant hypervariable regions of this gene; V3 and V6 as Pseudomonas aeruginosa MA01. The isolate was able to produce 12 g/l of a glycolipid-type biosurfactant and generally less efficient to emulsify vegetable oils compared to hydrocarbons and could emulsify corn and coconut oils more than 50%. However, emulsification index (E(24)) of different hydrocarbons including hexane, toluene, xylene, brake oil, kerosene and hexadecane was between 55.8% and 100%. The surface tension of pure water decreased gradually with increasing biosurfactant concentration to 32.5 mNm(-1) with critical micelle concentration (CMC) value of 10.1mg/l. Among all carbon substrates examined, vegetable oils were the most effective on biosurfactant production. Two glycolipid fractions were purified from the biosurfactant crude extracts, and FTIR and ES-MS were used to determine the structure of these compounds. The analysis indicated the presence of three major monorhamnolipid species: R(1)C(10)C(10), R(1)C(10)C(12:1), and R(1)C(10)C(12); as well as another three major dirhamnolipid species: R(2)C(10)C(10), R(2)C(10)C(12:1), and R(2)C(10)C(12). The strain sweep experiment for measuring the linear viscoelastic of biosurfactant showed that typical behavior characteristics of a weak viscoelastic gel, with storage modulus greater than loss modulus at all frequencies examined, both showing some frequency dependence.

Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws.

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels.

Structural characterization of a rhamnolipid-type biosurfactant produced by Pseudomonas aeruginosa MR01: enhancement of di-rhamnolipid proportion using gamma irradiation.

We previously reported that MR01, an indigenous strain of Pseudomonas aeruginosa, was able to produce a rhamnolipid-type biosurfactant. Here, we attempted to define the structural properties of this natural product. The analysis of the extracted biosurfactant by thin-layer chromatography (TLC) revealed the presence of two compounds corresponding to those of authentic mono- and di-rhamnolipid. The identity of two structurally distinguished rhamnolipids was confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Liquid chromatography/mass spectrometry (LC/MS) of extracted biosurfactant revealed up to seventeen different rhamnolipid congeners. Further quantification showed di-rhamnolipids as the major compound (77.2%), while monorhamnolipids comprising a smaller proportion (22.8%) of MR01 biosurfactant. Rha-Rha-C10-C10 was verified as the major component of the MR01 biosurfactant (35.93%). Cytotoxic activity of MR01 biosurfactant against human cancer Hela cells showed an excellent inhibitory effect of 5μg/ml. An isolated mutant strain (MR01-C) created by Gamma ray irradiation demonstrated more than one and a half-fold biosurfactant production and activity compared with the parent strain. Analysis of the biosurfactant produced by MR01-C showed the magnitude of di-rhamnolipids in the sample increased up to 88.6% (∼15% higher than control) and the quantity of Rha-Rha-C10-C10 increased to 52.08% (∼45% higher than control).

Enhanced phenol degradation by Pseudomonas sp. SA01: Gaining insight into the novel single and hybrid immobilizations

In this work, Pseudomonas sp. SA01 cells were immobilized in a series of singular and hybrid immobilization techniques to achieve enhanced phenol removal. The singular immobilization strategies consisted of various concentrations of alginate (2-4%) and pectin (3-5%), while the hybrid strategies incorporated polyvinyl alcohol (PVA)-alginate and glycerol-alginate beads and alginate-chitosan-alginate (ACA) capsules. Immobilization protected cells against phenol and resulted in remarkable reduction (65%) in degradation time by cells immobilized in either alginate (3%) beads, in a hybrid PVA-alginate beads, or in ACA capsules compared to freely suspended cells. Cells immobilized in PVA-alginate and ACA provided the best performance in experiments using elevated phenol concentrations, up to 2000 mg/L, with complete degradation of 2000 mg/L phenol after 100 and 110 h, respectively. Electron microscopy examination indicated that cell loading capacity was increased in PVA-alginate hybrid beads through reduced cell leakage, resulting in higher activity of PVA-alginate hybrid beads compared to all other immobilization methods.

Physicochemical characterization of a monorhamnolipid secreted by Pseudomonas aeruginosa MA01 in aqueous media. An experimental and molecular dynamics study.

Given the increasing interest in the characterization of new biosurfactants, in this work we have carried out a physicochemical study of a monorhamnolipid (monoRL) produced by Pseudomonas aeruginosa MA01 in aqueous media. The detailed knowledge of the physicochemical properties of these monoRL biosurfactant is of importance for the validation of this particular P. aeruginosa strain as a useful biosurfactant producer. A pKa value for monoRL of 5.9 was consistently obtained, as well as the indication that the presence of one or two rhamnose rings does not have a notorious influence on the pKa of the carboxyl group. The critical micelle concentration (cmc) of the negatively charged monoRL is dependent on the ionic strength, whereas that of the protonated form is not, whereas the charge of the polar head of monoRL has little effect on the surface area. Dynamic light scattering showed that in the vicinity of the cmc structures with an average diameter of 50 nm are present, whereas at concentrations well above the cmc the size increases to about 200 nm. Taken together our results show that monoRL presents a monomer-to-micelle transition, which depends on pH and ionic strength, similar to that described before for the diRL species. However the formation of lamellar vesicles described for diRL at pH 7.4, was not observed here. Molecular dynamics (MD) simulations yielded a similar value for the lateral diffusion coefficient of protonated anionic monoRL, indicating that the negative charge does not affect biosurfactant mobility in the monolayer surface. The radial distribution function value is slightly higher for the protonated monoRL; therefore the number of molecules located at a particular distance is somehow higher in the case of the protonated form. On the other hand, it is clearly obtained that the carboxylate group of the anionic form moves more inside the aqueous phase as compared to the carboxyl group of the protonated form. The results obtained correspond to the expected behaviour for a biosurfactant molecule in relation to the dependence of protonation state and micelle formation, and therefore the molecular dynamics simulation appears to describe properly our molecular systems.

Biochemical characterization of the decaprenyl diphosphate synthase of Rhodobacter sphaeroides for coenzyme Q10 production

Coenzyme Q10 (CoQ10), like other CoQs of various organisms, plays indispensable roles not only in energy generation but also in several other processes required for cells’ survival. In this study, a gene encoding for a decaprenyl diphosphate synthase (Rsdds) was cloned from Rhodobacter sphaeroides in Escherichia coli. The in vivo catalytic activity and product specificity of Rsdds were compared with those of a counterpart enzyme from Agrobacterium tumefaciens (Atdds) in E. coli as a heterologous host. In contrast with Atdds, Rsdds showed lower catalytic activity but higher product specificity for CoQ10 production, as indicated by the amount of CoQ9 formation. The higher product specificity of Rsdds was also confirmed by utilizing both Rsdds and Atdds for in vitro synthesis of polyprenyl diphosphates. Thin layer chromatography indicated that the Rsdds enzyme resulted in relatively much less solanesyl diphosphate formation. The purified Rsdds catalyzed the addition of isopentenyl diphosphate to dimethyl allyl diphosphate, geranyl diphosphate, ω,E,E-farnesyl diphosphate (FPP), and ω,E,E,E-geranylgeranyl diphosphate as priming substrates. The kinetic parameters of Vmax (pmol/min), KM (μM), kcat (1/min), and kcat/KM of the enzyme using FPP as the most appropriate substrate were determined to be 264.6, 13.1, 8.8, and 0.67, respectively.

In-depth diversity analysis of the bacterial community resident in the camel rumen.

The rumen compartment of the ruminant digestive tract is an enlarged fermentation chamber which houses a diverse collection of symbiotic microorganisms that provide the host animal with a remarkable ability to digest plant lignocellulosic materials. Characterization of the ruminal microbial community provides opportunities to improve animal food digestion efficiency, mitigate methane emission, and develop efficient fermentation systems to convert plant biomasses into biofuels. In this study, 16S rRNA gene amplicon pyrosequencing was applied in order to explore the structure of the bacterial community inhabiting the camel rumen. Using 76,333 quality-checked, chimera- and singleton-filtered reads, 4954 operational taxonomic units (OTUs) were identified at a 97% species level sequence identity. At the phylum level, more than 96% of the reads were affiliated to OTUs belonging to Bacteroidetes (51%), Firmicutes (31%), Proteobacteria (4.8%), Spirochaetes (3.5%), Fibrobacteres (3.1%), Verrucomicrobia (2.7%), and Tenericutes (0.95%). A total of 15% of the OTUs (746) that contained representative sequences from all major taxa were shared by all animals and they were considered as candidate members of the core camel rumen microbiome. Analysis of microbial composition through the solid and liquid fractions of rumen digesta revealed differential enrichment of members of Fibrobacter, Clostridium, Ruminococcus, and Treponema in the solid fraction, as well as members of Prevotella, Verrucomicrobia, Cyanobacteria, and Succinivibrio in the liquid fraction. The results clearly showed that the camel rumen microbiome was structurally similar but compositionally distinct from that of other ruminants, such as the cow. The unique characteristic of the camel rumen microbiome that differentiated it from those of other ruminants was the significant enrichment for cellulolytic bacteria.

RESPONSE SURFACE OPTIMIZATION OF BIOSURFACTANT PRODUCED BY Pseudomonas aeruginosa MA01 ISOLATED FROM SPOILED APPLES

A potent biosurfactant-producing bacterial strain isolated from spoiled apples was identified by 16S rRNA as Pseudomonas aeruginosa MA01. Compositional analysis revealed that the extracted biosurfactant was composed of high percentages of lipid (66%, w/w) and carbohydrate (32%, w/w). The surface tension of pure water decreased gradually with increasing biosurfactant concentration to 32.5 mN m−1 with critical micelle concentration (CMC) value of 10.1 mg L−1. The Fourier transform infrared spectrum of extracted biosurfactant confirmed the glycolipid nature of this natural product. Response surface methodology (RSM) was employed to optimize the biosynthesis medium for the production of MA01 biosurfactant. Nineteen carbon sources and 11 nitrogen sources were examined, with soybean oil and sodium nitrate being the most effective carbon and nitrogen sources on biosurfactant production, respectively. Among the organic nitrogen sources examined, yeast extract was necessary as a complementary nitrogen source for high production yield. Biosurfactant production at the optimum value of fermentation processing factor (15.68 g/L) was 29.5% higher than the biosurfactant concentration obtained before the RSM optimization (12.1 g/L). A central composite design algorithm was used to optimize the levels of key medium components, and it was concluded that two stages of optimization using RSM could increase biosurfactant production by 1.46 times, as compared to the values obtained before optimization.

Calcium carbonate precipitation by strain Bacillus licheniformis AK01, newly isolated from loamy soil: a promising alternative for sealing cement‐based materials

The relevant experiments were designed to determine the ability of indigenous bacterial strains isolated from limestone caves, mineral springs, and loamy soils to induce calcium carbonate precipitation. Among all isolates examined in this study, an efficient carbonate‐precipitating soil bacterium was selected from among the isolates and identified by 16S rRNA gene sequences as Bacillus licheniformis AK01. The ureolytic isolate was able to grow well on alkaline carbonate‐precipitation medium and precipitate calcium carbonate more than 1 g L−1. Fourier transform infrared (FTIR) spectroscopy, X‐ray diffraction (XRD) analyses, and scanning electron microscopy (SEM)/energy‐dispersive X‐ray spectroscopy (EDX) examinations were performed in order to confirm the presence of calcium carbonate in the precipitate and to determine which polymorphs were present. The selected isolate was determined to be an appropriate candidate for application in a surface treatment of cement‐based material to improve the properties of the mortar. Biodeposition of a layer of calcite on the surface of cement specimens resulted in filling in pore spaces. This could be an alternative method to improve the durability of the mortar. The kind of bacterial culture and medium composition had a profound impact on the resultant CaCO3 crystal morphology.

Lipid production and mixotrophic growth features of cyanobacterial strains isolated from various aquatic sites.

The present study was conducted to determine the potential of five cyanobacteria strains isolated from aquatic zones to induce lipid production. The phylogenetic affiliation of the isolates was determined by 16S rRNA gene sequencing. Amongst the isolates, an efficient cyanobacterium, Synechococcus sp. HS01 showing maximal biomass and lipid productivity, was selected for further studies. In order to compare lipid productivity, the HS01 strain was grown in different media to screen potential significant culture ingredients and to evaluate mixotrophic cultivation. Mixotrophic cultivation of the strain using ostrich oil as a carbon source resulted in the best lipid productivity. GC analysis of fatty acid methyl esters of the selected cyanobacterial strain grown in media supplemented with ostrich oil showed a high content of C16 (palmitoleic acid and palmitic acid) and C18 (linoleic acid, oleic acid and linolenic acid) fatty acids of 42.7 and 42.8 %, respectively. Transmission electron micrographs showed that the HS01 cells exhibited an elongated rod-shaped appearance, either isolated, paired, linearly connected or in small clusters. According to initial experiments, ostrich oil, NaNO3 and NaCl were recognized as potential essential nutrients and selected for optimization of media with the goal of maximizing lipid productivity. A culture optimization technique using the response surface method demonstrated a maximum lipid productivity of 56.5 mg l(-1) day(-1). This value was 2.82-fold higher than that for the control, and was achieved in medium containing 1.12 g l(-1) NaNO3, 1 % (v/v) ostrich oil and 0.09 % (w/v) NaCl.