Optimization of Degenerate PCR Conditions for Reducing Error Rates in Detection of PKS and NRPS Gene groups in Actinomycetes.

Background: The screen of Polyketide Synthase (PKS) and Nonribosomal Peptide Synthetase (NRPS) gene groups is a quick way to discover new therapeutic agents. However, errors in laboratory techniques cause a loss of touch with reality. This study aimed to evaluate the presence of PKS and NRPS gene groups in previously isolated strains by optimizing their specialized amplification by degenerate primers and indicating the evolutionary relationships with reference strains. Methods: PKS-I, II, and NRPS genes PCR amplification was performed using three degenerate primer sets for 22 actinomycete strains with antibacterial activity. Annealing temperature and the amount of template DNA and primers were optimized. PCR products of PKS-I, II, and NRPS from three strains were sequenced after TA cloning. Besides, strains with high antibacterial activity were identified by biochemical features and partial 16S rDNA sequencing and hypothetically classified by a phylogenetic tree. Results: High frequencies of PKS-I (86.4%), PKS-II (81.8%), and NRPS (95.4%) genes were found among the strains after optimization. Fourteen strains (64%) contained all of the genes, and 100% of strains had at least two genes. These numbers are pretty distinct in comparison with the previous researches. All of the sequenced strains were members of Streptomyces genus. Conclusion: Our research showed that degenerate PCR strongly depends on the variation of annealing temperature and primer concentration, resulting in an unexpected shift in PCR outputs. The sequencing results confirmed the optimized conditions for specialized PCR of PKS-I, PKS-II, and NRPS gene groups.

Biosynthesis of Calcite Nanocrystal by a Novel Polyextremophile Bhargavaea cecembensis-Related Strain Isolated from Sandy Soil.

Urease-producing bacteria are abundant in soils, which can precipitate calcium carbonate nanocrystals by enzymatic hydrolysis of urea in the presence of calcium ions. This process is known as microbially induced calcium carbonate precipitation (MICP), and it has received much attention in recent years as an eco-friendly technology. Therefore, the purpose of the present study was to isolate local extremophile bacterial strains capable of producing calcium carbonate. Among a total of 44 isolated urease-producing strains from sandy soils, one strain with a high level of urease activity (8.16 U/ml) and production of a large amount of calcium carbonate (410 mg/100 ml) was selected for further investigation. 16S rRNA gene sequencing showed that this strain had 99.66% sequence identity to Bhargavaea cecembensis. The SEM-EDX and XRD analyses indicated that irregular vaterite and aggregated nanocalcite were the dominant polymorphs produced by this strain. The size of these nanocalcite crystals ranged between 25 and 42 nm. The selected strain showed high levels of tolerance to different conditions of temperature, pH, and salinity. This strain grows at high temperatures up to 50 °C, alkaline pH (9-11), and high concentrations of NaCl (20-25% w/v). Flow cytometry analysis demonstrated 96% cell viability of the isolated strain after desiccation stress. Bhargavaea was first reported in 2009 as a new genus, and it belongs to the Firmicutes. So far, there has been no report on its MICP potential. The present study is the first one to report nanocrystal calcium carbonate precipitation in polyextremophile Bhargavaea cecembensis, which makes it a suitable candidate for bio-cementation under extreme circumstances.

Bacillales: From Taxonomy to Biotechnological and Industrial Perspectives.

For a long time, the genus Bacillus has been known and considered among the most applicable genera in several fields. Recent taxonomical developments resulted in the identification of more species in Bacillus-related genera, particularly in the order Bacillales (earlier heterotypic synonym: Caryophanales), with potential application for biotechnological and industrial purposes such as biofuels, bioactive agents, biopolymers, and enzymes. Therefore, a thorough understanding of the taxonomy, growth requirements and physiology, genomics, and metabolic pathways in the highly diverse bacterial order, Bacillales, will facilitate a more robust designing and sustainable production of strain lines relevant to a circular economy. This paper is focused principally on less-known genera and their potential in the order Bacillales for promising applications in the industry and addresses the taxonomical complexities of this order. Moreover, it emphasizes the biotechnological usage of some engineered strains of the order Bacillales. The elucidation of novel taxa, their metabolic pathways, and growth conditions would make it possible to drive industrial processes toward an upgraded functionality based on the microbial nature.

Enhancement of biomass and total carotenoid content of a UV-resistant strain of Dietzia maris in response to different carbon and nitrogen sources.

In the present study, the effect of various fermentation media on the production of carotenoid pigment in a radiation-resistant strain of Dietzia maris was reported. The biomass and pigment production of this strain was evaluated using various sources of carbon and nitrogen as well as different concentrations of whey medium. The antioxidant and cytotoxic activities of the extracted pigment were also determined using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays. The antibacterial activity of the carotenoid pigment was also evaluated. All carbon sources increased the pigment production of D. maris in the following order: glucose > raffinose = starch = xylose > sorbitol > sucrose. However, only glucose, xylose, and sorbitol significantly increased the microbial biomass as compared to the control. Moreover, all organic nitrogen sources and ammonium sulfate enhanced the pigment production of the studied strain by approximately 6-9 folds. The free radical scavenging capacity and FRAP of the D. maris carotenoid extract were reported as half-maximal effective concentration or EC50 = 3.30 mg/ml and EC50 =28.46 µg/ml, respectively. The maximum amount of biomass and carotenoid pigment produced by D. maris was obtained in the fermentation medium containing 1 g/l glucose and 1 g/l yeast extract (18 mg/l). This strain can be considered as a promising biocatalyst for the commercial production of natural carotenoids due to its antioxidant capacity and noncytotoxic activity.

Efficacy of polyextremophilic Aeribacillus pallidus on bioprocessing of beet vinasse derived from ethanol industries.

This work aimed to evaluate the applicability of Aeribacillus pallidus for the aerobic treatment of the concentrated beet vinasse with high chemical oxygen demand (COD 685 g.L-1) that is defined as an environmental pollutant. This bacterium is a polyextremophilic strain and grow aerobically up to 7.5% vinasse at high temperature (50 °C). In the bioreactor and under controlled conditions, A. pallidus reduced the soluble COD content of 5% vinasse up to 27% during 48 h and utilized glucose and glycerol, completely. Furthermore, a reduction of manganese, copper, aluminum, and nickel concentrations was observed in the treated vinasse with A. pallidus. The obtained results make this strain as an appropriate alternative to be used for the aerobic bioprocessing of the vinasse.

The Effect of Calcium/Magnesium Ratio on the Biomass Production of a Novel Thermoalkaliphilic Aeribacillus pallidus Strain with Highly Heat-Resistant Spores.

Hot springs are fascinating extreme environments for the isolation of polyextremophilic microorganisms with extraordinary characteristics. Since polyextremophilic bacterial growth are not as high as routine bacteria, the objective of this study was to investigate the effect of some environmental factors on biomass and metabolites productions in the newly isolated strain, from Larijan hot spring in Iran. The strain was identified as Aeribacillus pallidus Lhs-10 and deposited as CCUG 72355 and IBRC-M 11202 in Sweden and Iran, respectively. This thermoalkaliphilic strain can grow best at 50 °C, pH 8 and in the presence of 25 g/l NaCl. The physiological characterization of this strain show that [Ca/Mg] ratio affect its growth and biomass production with the best results obtained at the ratio of 2.5. Moreover, lactic and acetic acids production by this strain was affected by pH, aeration, and temperature, where a metabolic shift was detected from lactate to acetate production when the culture was aerated. Besides, its spores could tolerate heating at 80, 85, 90, 95 and 98 °C for 30 min without any reduction in the initial spore population, whereas D-value was defined 50 min at 98 °C. This newly lactic acid-producing strain of A. pallidus can be a promising strain that can be used in the harsh conditions in industrial processes.

An ancient plant for the synthesis of a novel carbon dot and its applications as an antibacterial agent and probe for sensing of an anti-cancer drug.

An innovative, facile, low- cost and one-pot hydrothermal synthesis was developed for preparing a high fluorescence carbon dots (CDs). In this report for the first time, Lawsonia inermis (Henna) plant as a carbon source was used to produce CDs without adding any chemical reagent and it was characterized by different techniques. The as-synthesized CDs exhibit high stability under various conditions and exceptionally solubility in hydrophilic solvents such as water and ethanol. In addition, the CDs were employed as a biocompatible probe for determination of methotrexate (MTX) in the range of 0.02 µmol L-1 to 18 µmol L-1 with a detection limit as 7 nmol L-1. Highly selective and sensitive determination of MTX was carried out through the fluorescence resonance energy transfer (FRET) mechanism. It is noteworthy that, the antibacterial studies of this CDs lead to interesting results which suggested Henna CDs kill Gram-positive and Gram-negative bacteria like an antibiotic drug. Also, compared with the henna plant, CDs show antibacterial properties in much lower concentrations.

Microbial diversity in the hypersaline Lake Meyghan, Iran.

Lake Meyghan is one of the largest and commercially most important salt lakes in Iran. Despite its inland location and high altitude, Lake Meyghan has a thalassohaline salt composition suggesting a marine origin. Inputs of fresh water by rivers and rainfall formed various basins characterized by different salinities. We analyzed the microbial community composition of three basins by isolation and culturing of microorganisms and by analysis of the metagenome. The basins that were investigated comprised a green ~50 g kg-1 salinity brine, a red ~180 g kg-1 salinity brine and a white ~300 g kg-1 salinity brine. Using different growth media, 57 strains of Bacteria and 48 strains of Archaea were isolated. Two bacterial isolates represent potential novel species with less than 96% 16S rRNA gene sequence identity to known species. Abundant isolates were also well represented in the metagenome. Bacteria dominated the low salinity brine, with Alteromonadales (Gammaproteobacteria) as a particularly important taxon, whereas the high salinity brines were dominated by haloarchaea. Although the brines of Lake Meyghan differ in geochemical composition, their ecosystem function appears largely conserved amongst each other while being driven by different microbial communities.

Natronoarchaeum persicum sp. nov., a haloarchaeon isolated from a hypersaline lake.

A novel halophilic archaeon, designated strain WIIAL99T, was isolated from Lake Meyghan, a hypersaline lake in Iran. Cells of strain WIIAL99T were non-motile, catalase-positive and oxidase-negative. Strain WIIAL99T required at least 2.5 M NaCl and 0.05 M MgCl2 for growth. Optimal growth was achieved at 3.5 M NaCl and 0.1 M MgCl2. The optimum pH and temperature for growth were pH 7.0 and 37-40 °C; it was able to grow at pH 6.0-8.5 and 20-55 °C. Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 8 % (w/v). The major polar lipids of strain WIIAL99T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, disulfated diglycosyl diether and one unidentified glycolipid. The DNA G+C content of strain WIIAL99T was 66.7 mol%. The closest relative was Natronoarchaeum rubrum JCM 17119T with 98.2 % similarity in the orthologous 16S rRNA gene sequence. Analysis of 16S rRNA and rpoB' gene sequences indicated that strain WIIAL99T is a member of the genus Natronoarchaeum in the family Halobacteriaceae and forms a distinct cluster. It was concluded that strain WIIAL99T (=IBRC-M 11062T=LMG 29814T) represents a novel species of the genus Natronoarchaeum, for which the name Natronoarchaeum persicum sp. nov. is proposed.

Efficient Media for High Lipase Production: One Variable at a Time Approach.

BACKGROUND: Lipase enzymes have applications in a wide range of industries. A crucial determining factor of industrial prices of these enzymes is the culture media composition that is constantly under review by researchers. In this work, for maximum lipase production by Bacillus sp. ZR-5, culture media compositions were optimized using ″one variable at a time″ strategy. METHODS: For this purpose, the culture medium parameters such as low and high cost carbon and nitrogen sources, substrates and incubation times were evaluated. RESULTS: Maximum lipase activity was achieved after 24 hr of incubation with 1.5% of glucose syrup (1600±69.1 u/mg), 1% of fish powder (1238±36.7 u/mg) and olive oil (1407±2.1 u/mg) as low cost carbon and nitrogen sources and substrate, respectively. CONCLUSION: Our results show a significant increase in lipase activity with usage of low cost sources; this could help in reducing the media prices for industrial application of lipase enzyme.

Enhancement of production and activity of alkaline zinc metalloprotease from Salinivibrio proteolyticus using low intensity direct electric current and zinc nanoparticles.

OBJECTIVES: To improve the production and activity of an alkaline zinc metalloprotease from Salinivibrio proteolyticus in response to ZnSO4 (ionic and nanoparticle forms) and low intensity direct electric current (LIDC). RESULTS: A DC of 50 µA for 10 min increased enzyme production from 35 to 53 U ml(-1) when applied to the stationary phase bacterial cells. Zn(2+) improved enzyme production better than zinc nanoparticles (52 vs. 43.5 U ml(-1)). Zinc nanoparticles (0.5 mM) added to an enzyme reaction mixture containing casein (0.65 %) and 20 mM Tris/HCl buffer (pH 8) improved enzyme activity more than Zn(2+) (42 vs. 36 U ml(-1)). CONCLUSION: LIDC exposure (50 µA, 10 min) to the stationary phase bacterial cells increases metalloprotease production in Salinivibrio. A low concentration of zinc nanoparticles (0.5 mM) increases maximum enzyme activity.

Heterotrophic Bioleaching of Sulfur, Iron, and Silicon Impurities from Coal by Fusarium oxysporum FE and Exophiala spinifera FM with Growing and Resting Cells.

Coal is the most abundant fossil fuel containing sulfur and other elements which promote environmental pollution after burning. Also the silicon impurities make the transportation of coal expensive. In this research, two isolated fungi from oil contaminated soil with accessory number KF554100 (Fusarium oxysporum FE) and KC925672 (Exophiala spinifera FM) were used for heterotrophic biological leaching of coal. The leaching were detected by FTIR, CHNS, XRF analyzer and compared with iron and sulfate released in the supernatant. The results showed that E. spinifera FM produced more acidic metabolites in growing cells, promoting the iron and sulfate ions removal while resting cells of F. oxysporum FE enhanced the removal of aromatic sulfur. XRF analysis showed that the resting cells of E. spinifera FM proceeded maximum leaching for iron and silicon (48.8, 43.2 %, respectively). CHNS analysis demonstrated that 34.21 % of sulfur leaching was due to the activities of resting cells of F. oxysporum FE. Also F. oxysporum FE removed organic sulfur more than E. spinifera FM in both growing and resting cells. FTIR data showed that both fungi had the ability to remove pyrite and quartz from coal. These data indicated that inoculations of these fungi to the coal are cheap and impurity removals were faster than autotrophic bacteria. Also due to the removal of dibenzothiophene, pyrite, and quartz, we speculated that they are excellent candidates for bioleaching of coal, oil, and gas.

Isolation of Dickeya dadantii strains from potato disease and biocontrol by their bacteriophages.

One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other parts of the potato and other plants of the Solanaceae family. The application of restricted host range bacteriophages as biocontrol agents has recently gained widespread interest. This study purposed to isolate the infectious agent of the potato and evaluate its biocontrol by bacteriophages. Two phytopathogenic strains were isolated from infected potatoes, identified based on biochemical and 16S rRNA gene sequencing, and submitted to GenBank as D. dadantii strain pis3 (accession no. HQ423668) and D. dadantii strain sip4 (accession no. HQ423669). Their bacteriophages were isolated from Caspian Sea water by enriching the water filtrate with D. dadantii strains as hosts using spot or overlay methods. On the basis of morphotypes, the isolated bacteriophages were identified as members of the Myoviridae and Siphoviridae families and could inhibit the growth of antibiotic resistant D. dadantii strains in culture medium. Moreover, in Dickeya infected plants treated with bacteriophage, no disease progression was detected. No significant difference was seen between phage-treated and control plants. Thus, isolated bacteriophages can be suggested for the biocontrol of plant disease caused by Dickeya strains.

Isolation and Characterization of a New Thermoalkalophilic Lipase from Soil Bacteria.

Lipases are diversified enzymes in their properties and substrate specificity, which make them attractive tools for various industrial applications. In this study, an alkalinethermostable lipase producing bacteria were isolated from soil of different regions of Isfahan province (Iran) and its lipase was purified by ammonium sulfate precipitation and ion exchange chromatography. To select a thermoalkalophil lipase producing bacterium, Rhodamine B and Horikoshi media were used and the strain that can grow at 45° Cwas selected. The isolated strain was identified using microbial and biochemical tests. One strain showed an orange colored zone on plate and grew on Horikoshi plate. Microbial and biochemical tests showed that the isolated strain was Bacillus subtilis, a Gram positive rod. In PCR, an expected band was obtained with about 371 bp. The activity of the purified lipase was 10.2 folds that of the standard enzyme using ammonium sulfate precipitation and ion exchange chromatography. The molecular weight of lipase determined by SDS-PAGE electrophoresis, was 21 and 35 KDa. Existence of two bands in SDS-PAGE electrophoresis and low amount of obtained purified enzyme highlights the necessity of optimization of purification and concentrating process.

Isolation of Dickeya dadantii strains from potato disease and biocontrol by their bacteriophages

One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other parts of the potato and other plants of the Solanaceae family. The application of restricted host range bacteriophages as biocontrol agents has recently gained widespread interest. This study purposed to isolate the infectious agent of the potato and evaluate its biocontrol by bacteriophages. Two phytopathogenic strains were isolated from infected potatoes, identified based on biochemical and 16S rRNA gene sequencing, and submitted to GenBank as D. dadantii strain pis3 (accession no. HQ423668) and D. dadantii strain sip4 (accession no. HQ423669). Their bacteriophages were isolated from Caspian Sea water by enriching the water filtrate with D. dadantii strains as hosts using spot or overlay methods. On the basis of morphotypes, the isolated bacteriophages were identified as members of the Myoviridae and Siphoviridae families and could inhibit the growth of antibiotic resistant D. dadantii strains in culture medium. Moreover, in Dickeya infected plants treated with bacteriophage, no disease progression was detected. No significant difference was seen between phage-treated and control plants. Thus, isolated bacteriophages can be suggested for the biocontrol of plant disease caused by Dickeya strains.

Sulfur Removal from Dibenzothiophene by Newly Isolated Paenibacillus validus Strain PD2 and Process Optimization in Aqueous and Biphasic (Model-Oil) Systems.

Dibenzothiophene (DBT) is an organic sulfur compound which remains in oil after hydrodesulfurization (HDS) process and can be removed by biodesulfurization (BDS). A new strain of Paenibacillus validus (strain PD2) was isolated from oil contaminated soils that is able to desulfurize DBT. HPLC analysis and Gibbs assay showed that this strain was capable to convert DBT to 2-Hydroxybiphenyl (2-HBP) as final product. The presence of dszC gene confirmed that DBT desulfurization occurred through the 4S pathway. Maximum growth and the highest induction in dsz operon obtained in the presence of dimethyl sulfoxide (DMSO) as sole sulfur source. DBT concentration, temperature and pH were optimized statistically for growing and resting cells by using Response Surface Methodology (RSM). All parameters in growing cells had a significant effect on 2-HBP production during BDS of DBT by P. validus PD2, although in resting cells temperature in range of 20-40 degrees C was not a significant factor. Maximum BDS for growing cells was obtained at 0.41 mM DBT concentration, pH 6.92 and temperature 31.23 degrees C. For resting cells, optimum pH, temperature and DBT concentration were 6.62, 27.73 degrees C and 7.86 mM respectively. The results of this study showed that high concentrations of DBT could be desulfurized by P. validus strain PD2 in model-oil. Thus, the isolated strain could be introduced as a proper candidate for biodesulfurization of organic sulfur in the oil industry.

A novel metabolite (1,3-benzenediol, 5-hexyl) production by Exophiala spinifera strain FM through dibenzothiophene desulfurization.

Sulfur dioxide which is released from petroleum oil combustion causes pollution over the atmosphere and the soil. Biodesulfurization can be used as a complementary method of hydrodesulfurization, the common method of petroleum desulfurization in refineries. Many studies have been carried out to develop biological desulfurization of dibenzothiophene (DBT) with bacterial biocatalysts. However, fungi are capable to metabolize a wide range of aromatic hydrocarbons through cytochrome P450 and their extracellular enzymes. The aim of the present work was isolation and identification of fungi biocatalysts capable for DBT utilization as sulfur source and production of novel metabolites. DBT consumption and the related produced metabolites were analyzed by HPLC and GC-MS respectively. One of the isolated fungi that could utilize DBT as sole sulfur source was identified by both traditional and molecular experiments and registered in NCBI as Exophiala spinifera FM strain (accession no. KC952672). This strain could desulfurize 99 % of DBT (0.3 mM) as sulfur source by co-metabolism reaction with other carbon sources through the same pathway as 4S and produced 2-hydroxy biphenyl (2-HBP) during 7 days of incubation at 30 °C and 180 rpm shaking. However, the isolate was able to transform 2-HBP to 1,3-benzenediol, 5-hexyl. While biphenyl compounds are toxic to leaving cells, biotransformation of them can reduce their toxicity and the fungi will be more tolerant to the final product. These data are the first report about the desulfurization of DBT comparable to 4S-pathway and production of innovative metabolite by E. spinifera FM strain.

Improvement of Biodesulfurization Rate of Alginate Immobilized Rhodococcus erythropolis R1.

BACKGROUND: Sulfur oxides released from the burning of oil causes severe environmental pollution. The sulfur can be removed via the 4S pathway in biodesulfurization (BDS). Immobilization approaches have been developed to prevent cell contamination of oil during the BDS process. OBJECTIVES: The encapsulation of Rhodococcus erythropolis R1 in calcium alginate beads was studied in order to enhance conversion of dibenzothiophene (DBT) to 2-hydroxy biphenyl (2-HBP) as the final product. Also the effect of different factors on the BDS process was investigated. MATERIALS AND METHODS: Calcium alginate capsules were prepared using peristaltic pumps with different needle sizes to control the beads sizes. Scanning electron microscopy and flow cytometry methods were used to study the distribution and viability of encapsulated cells, respectively. Two non-ionic surfactants and also nano Ƴ-Al2O3were used with the ratio of 0.5% (v/v) and 1:5 (v/v) respectively to investigate their BDS efficiency. In addition, the effect of different bead sizes and different concentrations of sodium alginate in BDS activity was studied. RESULTS: The 2% (w/v) sodium alginate beads with 1.5mm size were found to be the optimum for beads stability and efficient 2-HBP production. The viability of encapsulated cells decreased by 12% after 20 h of desulfurization, compared to free cells. Adding the non-ionic surfactants markedly enhanced the rate of BDS, because of increasing mass transfer of DBT to the gel matrix. In addition, Span 80 was more effective than Tween 80. The nanoƳ-Al2O3 particles could increase BDS rate by up to two-folds greater than that of the control beads. CONCLUSIONS: The nano Ƴ-Al2O3 can improve the immobilized biocatalyst for excellent efficiency of DBT desulfurization. Also the BDS activity can be enhanced by setting the other explained factors at optimum levels.

Isolation and characterization of novel thermophilic lipase-secreting bacteria

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.