فهرست مطالب mohammad reza soudi

The genus Xanthomonas belongs to the Xanthomonadaceae family and the Gammaproteobacteria class, which includes short rod-shaped gram-negative bacteria that cause disease in more than 400 different plant hosts and are the most important pathogen in a wide range of plants, which results in a decrease in productivity in the agricultural industry.Pathogenic bacteria in plants including Xanthomonas campestris pv. campestris are one of the most important factors that reduce the productivity of agricultural products and are responsible for major economic losses in the agricultural industry. This bacterial strain is pathogenic in a wide range of plants such as rice, wheat, citrus fruits, tomatoes, peppers, cabbage, melons, bananas, and seeds such as beans. The methods of dealing with the disease that has been used against them so far involve environmental damage, the accumulation of toxins in the soil, and bacterial resistance. Therefore, it seems that more effective methods are needed. Bacteriophages, Viruses infect bacteria, which are exclusive to their host attack, and as new, safe, and effective inhibitory agents against plant pathogenic bacteria, they have been much considered in recent studies.In the studies conducted on isolated phages effective against Xanthomonas bacteria, promising results have been obtained in the field of plant disease management in laboratory and field conditions. In this regard, the identification and complete knowledge of the biological characteristics of phages effective against this bacterium is very crucial for the development of effective biological control products.

In the present study, isolation and identification of lytic bacteriophage effective on strains of Xanthomonas campestris bacteria were investigated to inhibit two forms of planktonic and biofilm bacteria so that it can be used in the future to fight the contamination of agricultural products with this pathogenic bacterium. Under sterile conditions and with a sterile loop, a single plaque was completely removed from the selective plate containing phage plaques and autoclaved in 5 ml of YMB medium and 10 μl of fresh suspension of the bacterial strain X. campestris DSM 1706 was added and incubated for 24 hours in the incubator was incubated at a temperature of 28 degrees Celsius and aeration at 150 rpm. Then the solution was centrifuged for 10 minutes at a speed of 6000 rpm and the supernatant was filtered with a 0.45 micron needle filter.Lytic phage is effective on the strain Xanthomonas campestris pv. campestris DSM1706. It was separated from the water of the Karun River by the two-layer agar method. The phage host range to the staining method was checked. Then, phage morphology through transmission electron microscopy (TEM) imaging, phage stability against pH and different temperatures, one-step proliferation curve, and the effect of phage on removing and inhibiting bacterial biofilm were investigated.

In this study, clear phage plaques, caused by the proliferation of lysing phages of the desired strain, were observed. Phage isolated on the morphological characteristic was based on the tectonic family. In addition to the strain Xanthomonas campestris pv. campestris DSM 1706, it had a lytic effect on three other pathogenic isolates obtained from infected cabbage fields including SAM 4209, SAM 4211, and SAM 4212. This phage had high stability in the temperature range of 20 °C to 50 °C and was inactivated at -70 °C. It also had good lytic activity in the range of pH 5 to 10 and became inactive at pH 3. According to the one-step proliferation curve, the phage proliferation cycle lasts about 70 minutes which includes a 25–30-minute incubation phase and a 40-minute phage release phase. Phage showed an 86% inhibitory effect and 93% elimination effect on the biofilm of this bacterium.In the present study, phage stability against different temperatures and acidity was studied as two main factors. The results showed that the phage has good lytic activity in acidity between 5 and 9, its performance decreases in alkaline acidity more than 9 and it becomes inactive in acidity 3 and does not show any lytic activity against the target bacteria. The best performance and the highest lytic activity were observed at an acidity of about 7.

Since the first discovery of phages in this study, it has been proven that phages as a biological control strategy are promising alternatives with many advantages for agricultural chemicals and antibiotics. Based on the results of this research, the obtained lytic phage has stability in the spectrum in laboratory conditions (a wide range of pH and temperatures) and was able to lyse the host bacteria in planktonic cell form and inhibit and remove the bacterial biofilm with a high percentage. As a result, it can be a biological agent with high potential in controlling plant diseases and replacing combat methods as a common and suitable candidate for phage therapy.In several studies in this field, it has been shown that the use of different compounds mixed with solutions containing phage increases the persistence of phage on leaf surfaces. For example, in one study, the use of skim milk and this combination led to improved control of tomato bacterial spot disease compared to standard treatment with chemical bactericides.In line with this study, it is suggested to investigate the effect of phage on other pathogenic strains of Xanthomonas bacteria and also on their biofilm in different ways. Among other things, specific lytic bacteriophages of Xanthomonas bacteria can be used individually or in combination with other isolated bacteriophages along with antibiotics or other control agents such as copper compounds in preventive and therapeutic measures as phage therapy against bacterial infections.

The widespread use of chemical fertilizers became common by coming the Haber-Bosch reaction product to the market in the second decade of the twentieth century, and thereafter the agricultural industry flourished. Nowadays, increasing usage of chemicals has caused adverse and harmful consequences against the environment and also increased costs of production of crop plants. The use of biofertilizers, which potentially solubilizes phosphorus compounds in the agricultural soil, is considered as an environmental friendly option and a superior alternative to further usage of chemical fertilizers. New collections of phosphate-solubilizing bacteria with a vast biodiversity are required to be used for greater varieties of crop plants in more different climates. The aim of this study was verification and characterization of some active phosphate-solubilizing bacteria isolated from several areas of wheat arable soils located in Yazd province.

In this study, soil microorganisms were screened and all the isolates were examined in PVK agar culture medium for halo formation and then phosphate solubilization index and efficiency were calculated. Phosphate release rate in liquid culture medium was measured in µg/ml. Nitrogen fixation test using two carbon sources (glucose and sucrose) was evaluated comparatively. All the isolates were examined for tolerance to various environmental conditions such as temperature, salinity and pH. Finally, the production of several types of organic acids by the isolates was examined using thin layer chromatography.

Among the bacteria, 10 isolates created a halo zone in culture medium containing calcium phosphate and showed growth and discoloration in nitrogen-free medium with bromothymol blue reagent. Five isolates out of 10 showed the highest efficiency in phosphate solubilization (equal to or more than 200% Z/C) and 5 isolates showed the highest phosphate solubility index (between 2- 4). The highest index belonged to YC isolate (4.1), while statistical analysis showed no significant difference between the means of solubility efficiency of different isolates. The quantitative study showed that the amount of phosphate released for 4 superior isolates was 150.25-203.91 µg/ml. Statistical analysis shows that among the 10 strains tested, YS and YG showed the highest efficiency with the release of 203.9 and 194.8 μg/l phosphate, respectively. These two strains were not significantly different from YK and YC isolates. Isolates YX, YA, YP, YH, YJ and YI also showed the least ability at the same statistical level. All the isolates were able to fix nitrogen in the presence of glucose and 4 isolates were able to fix nitrogen in the presence of sucrose as two different carbon sources. Experiments carried out to show the resistance of the isolates to environmental factors and indicated that almost all isolates can grow adequately in the range of acidic to alkaline pH (5-9) and temperatures of 25-37 ºC. The isolates were also able to tolerate salinity up to 10% (w/v). The use of thin layer chromatography confirmed the excretion of organic acids such as citric acid and oxalic acid by the examined bacteria.

The present study indicates that the isolated microorganisms with high phosphate solubilizing ability show significant potential as a suitable substitute to chemical fertilizers with harmful side effects.

Microbial phytase is one of the most widely used enzymes in food industries like cattle, poultry, and aquaculture food. Therefore, understanding the kinetic properties of the enzyme is very important to evaluate and predict its behavior in the digestive system of livestock. Working on phytase is one of the most challenging experiments because of some problems, including free inorganic phosphate (FIP) impurity in phytate (substrate) and interference reaction of the reagent with both phosphates (product and phytate impurity).

In the present study, FIP impurity of phytate was removed, and then it was shown that the substrate (phytate) has a dual role in enzyme kinetics: substrate and activator.

phytate impurity was decreased by two-step recrystallization prior to the enzyme assay. The impurity removal was estimated by the ISO30024:2009 method and confirmed by Fourier-transform infrared (FTIR) spectroscopy. The kinetic behavior of phytase activity was evaluated using the purified phytate as substrate by non-Michaelis-Menten analysis, including Eadie-Hofstee, Clearance, and Hill plots. The possibility of an allosteric site on phytase was assessed by molecular docking.

The results showed a 97.2% decrease in FIP due to recrystallization. The phytase saturation curve had a sigmoidal appearance, and Lineweaver-Burk plot with a negative y-intercept indicated the positive homotropic effect of the substrate on the enzyme activity. A right-side concavity of Eadie-Hofstee plot confirmed it. Hill coefficient was calculated to be 2.26. Molecular docking also showed that Escherichia coli phytase molecule has another binding site for phytate very close to the active site, called “allosteric site”.

The observations strongly propose the existence of an intrinsic molecular mechanism in Escherichia coli phytase molecules to be promoted for more activity by its substrate, phytate (positive homotropic allosteric effect). In silico analysis showed that phytate binding to the allosteric site caused new substrate-mediated inter-domain interactions, which seems to lead to a more active conformation of phytase. Our results provide a strong basis for animal feed development strategies, especially in the case of poultry food and supplements, regarding a short food passage time in their gastrointestinal tract and variable concentration of phytate along with it. Additionally, the results strengthen our understanding of phytase auto-activation as well as allosteric regulation of monomeric proteins in general.

Fungal extracts have received increased attention due to their great medicinal applications including antitumor, immune-modulating, antioxidant, radical scavenging, antiviral, antibacterial, antifungal and detoxificating properties.

This study is the first report on a novel bioactive compound, namely Childinan SF-2 which was isolated from soil ascomycete fungus. The significant antibacterial, antioxidant and cytotoxic properties of the extract may lead to development of novel, safe and useful substances.

The isolate was identified on the basis of molecular approach. Spore suspension was inoculated in the culture medium and the bioactive compound was isolated from the viscous fermented broth via ethanol precipitation of the extracellular compound. The basic chemical composition of the extract including protein, carbohydrate, sulfate radical and uronic acid content were measured. FTIR (Fourier-transform infrared spectroscopy) and GC–MS (Gas chromatography–mass spectrometry) analysis were used for further structural characterization. The extract was utilized for treatment of AGS and MDA cell lines to assess the cell cycle and apoptosis. The antioxidant activity was examined using DPPH, hydroxyl radicals scavenging, β-carotene bleaching inhibition and ferric reducing power assay methods. The extract was tested for evaluation of antibacterial activity using different Gram-positive and Gram-negative bacterial strains

The fungal isolate was identified as the new strain Daldinia childiae SF-2. Initial biochemical characterization of the extract showed that the fungal biopolymer was composed of total sugars, protein, uronic acids and sulfated groups with values of 91.6%, 2.15%, 2.25% and 1.05% (w/w), respectively. FTIR and GC–MS analysis revealed that Childinan SF-2 might be mainly constructed from D-glucose, D-mannitol and D-galactofuranose. The in vitro experiments revealed that Childinan SF-2 enhanced the percentage of necrosis and apoptosis of cancer cells and blocked the cell cycle progression as shown by flowcytometry. Childinan SF-2 represented a considerable antioxidant and antibacterial activity.

These results indicated that Childinan SF-2 can serve as a potential source in medicinal applications.

Antibiotic-resistant bacteria are a major threat to global health. Older antibiotics have become more or less ineffective as a result of widespread microbial resistance and an urgent need has emerged for the development of new antimicrobial strategies. Acidocin 4356 is a novel antimicrobial bacteriocin peptide produced by Lactobacillus acidophilus ATCC 4356 and capable of confronting the Pseudomonas aeruginosa ATCC 27853 infection challenges. According to our previous studies, the production of Acidocin 4356 is in parallel with cellular biomass production.

Given the costly production of Acidocin 4356, the development of a beneficial approach for increasing productivity of the cellular biomass has been targeted in the lab-scale fermenter for scale-up production of this bacteriocin. Therefore, in this study, we developed an inexpensive optimal culture medium based on the whey feedstock, evaluating this medium for scaling-up of the bacteriocin production from flask to fermenter.

In the first step, the optimization of the process parameters and medium components was carried out using the Plackett-Burman (PB) design and Response surface methodology (RSM) in flask culture. After optimization of the medium, bacteriocin production in the optimum culture medium was compared with de Man, Rogosa and Sharpe (MRS) medium by analyzing the intensity of the peptide band. Intensity analysis has been conducted on the PAGE band of the peptide using Image J software. Finally, the scale- up of bacteriocin production in the optimum culture medium was evaluated by batch fermentation in a 3-liter fermenter.

In this study, a medium containing whey (40 g.L-1) and sodium acetate (5 g.L-1) was used as basal medium, and the effect of other factors were then evaluated. According to the PB design, three factors of peptone concentration, yeast extract concentrations and cultivation temperature were selected as the most effective factors which improve the growth of L. acidophilus. The condition providing the highest growth capacity for bacteriocin production were predicted based on the results of RSM as following: temperature 40 ° C, yeast (4 g.L-1), and peptone (8 g.L-1). Finally, the dry cell weight was obtained after incubation for 12 h as 2.25 g.L-1. Comparison of cell growth and bacteriocin production between MRS medium and optimized medium confirmed the efficacy of these optimal conditions for the cost-effective production of Acidocin 4356 in the flask. Besides, the scale- up of bacteriocin production has made under optimal condition in the 3-L fermenter.

In this study, for the first time, scale- up production of Acidocin 4356 was presented by using a low-cost method based on whey feedstock to tackle P. aeruginosa infections.

Polyethylene (PE) is one of the most abundant plastic wastes which accumulates in marine and terrestrial environments. As microbial degradation has been a promising approach for the bioremediation of polluted environments, identification of the microbial community profile where these pollutants accumulate, has recently been in focus. We have investigated the taxonomic and functional characteristics of polyethylene- degrading microorganisms in a plastic waste recycling site in Tehran, Iran. We have analyzed and compared a 16S rRNA dataset from this study with 15 datasets from 4 diverse plastic and oil polluted habitats to identify and evaluate bacterial communities involved in bioremediation. Our findings reveal that Proteobacteria, Actinobacteria, Acidobacteria and Cloroflexi were the dominant phyla and Actinobacteria, Alphaproteobacteria, Gammaproteobacteria and Acidimicrobia were dominant classes in these samples. The most dominant Kegg Orthology associated with PE bioremediation in these samples are related to peroxidases, alcohol dehydrogenases, monooxygenases and dioxygenases. Long-term presence of contaminants in soil could lead to changes in bacterial phyla abundance, resulting in metabolic adaptations to optimize biological activity and waste management in a diverse group of bacteria.

Nowadays, heavy metals pollution is one of the most important environmental problems. Due to the high cost of common refining methods, the use of microbial biomass is recommended to clean up heavy metals. The aim of this study was the evaluation of interactions of Rhodoterula toruloides strain IR-1395 with mercury.

In this study, the growth rate and interactions of strain IR-1395 with mercury in Sucrose Broth medium were investigated and the amount of mercury accumulated was measured using the Spectrophotometry method. Then, in order to determine the best biosorption conditions, the effect of parameters such as pH, mercury concentration, biomass concentration, contact time and temperature were measured. Also, the biosorption was investigated by treated cells with 2, 4 dinitrophenol and autoclave. Finally, the biosorbent was characterized by Scanning Electron Microscope with Energy Dispersive X-Ray Analysis.

Studies showed that the maximum growth rate of strain IR-1395 was after 36h and then it enters the stationary phase. Also, this strain was able to the biosorption and bioaccumulation (61.86%) and 10.17% biovolatilization of mercury from the medium at the concentration of 10 mg/l of chloride mercury during seven days. In study of factors affecting on the mercury biosorption, it is indicated that the optimal pH was 4. The highest biosorption was observed at 300 mg /l of mercury in 10 minutes at 15 ˚C. Mercury adsorption using treated biomass by autoclaved and 2,4 dinitrophenoltreatment has reduced than control samples. Finally, the results of the EDX confirmed the biosorption of mercury by this yeast.

By study of the various interactions of strain IR-1395 with mercury and its potential in biosorption, it was found that this strain is a valuable microorganism in mercury bioremediation processes from contaminated aquatic environments.

Poly(3-hydroxybutyrate) (PHB) is a well-known biodegradable polymer produced by some microorganisms and can be a suitable alternative for petrochemical plastics. PHB synthase encoded by phbC gene is the main enzyme in PHB biosynthesis pathway in Ralstonia eutropha. The aim of current study was the transformation of R. eutropha PTCC 1615 with its own phbC gene and evaluation of the overexpression effect on PHB accumulation.

DNA fragment including phbC gene and its promoter and terminator regions, was isolated from R. eutropha PTCC 1615, inserted into pET28a(+) vector, and transferred to the competent bacteria using calcium chloride and heat shock method. The effect of the cloned gene expression on PHB production was investigated with absorption of crotonic acid produced through PHB dehydration. Statistical analyses were carried out by SPSS software.

PHB content of cells of the engineered strain was 1.4 times more than that of the native bacteria. This significant difference can be an important finding for improvement of biopolymer production.

Overexpression of phbC, the critical gene in PHB biosynthesis pathway, in R. eutropha PTCC 1615 had considerable effect on PHB accumulation.

Polycyclic aromatic hydrocarbons are one of the most important environmental pollutants. Bioremediation using microorganisms is a cost-effective and safe method for the removal or conversion of these pollutants to less toxic substances. This study aimed to isolate and introduce fluorene-degrading microorganisms from the southern coast of the Caspian Sea. The mixed microbial culture enrichment and isolation was done in salt-based culture medium containing fluorene. The qualitative analysis of fluorene degradation in the solid basal salt medium was investigated. The rate of fluorene removal by the isolated mixed microbial culture was also determined using gas chromatography in a liquid salt base medium. The molecular identification of the fungal and bacterial isolates was performed using the sequential analysis of the ITS protected region and 16S rRNA sequencing, respectively. The mixed microbial culture including bacterial isolates (belonging to the genus Pseudomonas, Acromobacter, Chryseobacterium, Microbacterium, and Rhodococcus) and fungal isolate (belonging to the genus Fusarium) was enriched and isolated. Chromatographic analysis showed that the mixed microbial culture was able to degrade 87% of fluorene (200 mg / l ) in a basal salt medium at 30 °C, pH 7 and 7 days of incubation. According to the results, the mixed microbial can remove a large amount of fluorene from the basal salt medium under the mentioned conditions and it is possible that under a similar situation, it can remove a large amount of fluorene from the contaminated area through bioremediation.

Based on information about the types and numbers of microorganisms in the surface soil samples, the distribution range of underlying oil and gas reservoirs can be judged. In the present study, methane oxidizing bacteria were isolated and characterized from oil contaminated soils in Khuzestan, Iran and their growth was optimized in the presence of methane as sole carbon and energy sources. 

Materials & Methods:

Bacteria were isolated in nitrate mineral salt (NMS) medium in the presence of 50% air and 50% methane. The cultures were incubated on a shaker for 10 days at 30°C in dark place, and every two days, the gas mixture was replaced. The isolated bacteria were characterized based on biochemical and molecular identification tests. Then the optimum growth conditions was detected in different pH values and incubation temperatures in NMS medium containing methane.

Three Gram-negative rods were isolated from soil samples that were able to grow in isolation condition. The isolates were characterized as Achromobacter and Sphingomonas spp. The strains could also grow in the NMS medium with a high methanol concentration (3%). The optimal pH and temperature for the isolates were 7.4 and 30°C respectively.

Methanotrophic strains that were isolated in the present study were able to grow and oxidize methane in high ranges of temperature and pH and can be proposed for the removal of mono-carbon compounds such as methane and as biological detectors for prospecting for oil and gas reservoirs

Xanthomonas campestris is a biopolymer producing gram negative bacterium. Production of xanthan biopolymer can be affected by different extrinsic factors as well as surfactants. Hitherto, effects of nonionic surfactants on xanthan production have been studied in a limited number of articles.
In the present study, nonionic surfactants were used to pursue their effects on improvement of xanthan production. Moreover, a number of cellular consequences upon the treatment were investigated with impacts on gum production.
Effects of different nonionic surfactants (Tween 20, Tween 80 and Triton X100) on xanthan production and Xanthomonas campestris cells were assessed by ultramicroscopy (SEM), changes in culture turbidity, leakage of sugars and ATP, and quality of xanthan (i.e. pyruvate content and determination of polymer molecular weight).
The nonionic surfactant Tween 20 increased ATP (3.2 folds) and sugar leakage (3.1 folds). Furthermore, they caused cell shape alteration. Tween 80 improved both xanthan production (11 g.L-1) and viscosity of the product (1368 cP), while the total biomass remained unchanged (2.2 g.L-1). Molecular weight of xanthan was enhanced (from 23 to 59 million Da). Toxic effect of 5% (v/v) Triton X 100 decreased the turbidity of culture to 120 NTU and total biomass was diminished to 1 g.L-1. Tween 20 caused the loss of ATP and sugar leakage and led to lower xanthan production. It had no effect on biomass content.
In general, amounts of surfactants that bacterial cells can tolerate seem to be helpful in substrate and metabolite transportation, and enzyme activities involved in xanthan biosynthesis and release. Surfactants induce harsh damages to cell barriers, preventing the growth and adversely affecting quantity and quality of xanthan gum.

A number of microorganisms and their enzymes have been reported as xanthan depolymerizers.Paenibacillus species are well-known polysaccharide hydrolyzing bacteria. However, Paenibacillus alginolyticus and Paenibacillus sp. XD are the only species in the genus which are now known to degrade xanthan.
Complete biodegradation of the xanthan exopolysaccharide is a rarely found capability among microorganisms. The aim of this study is to survey xanthanase producing bacteria with an appropriate bioactivity for the biopolymer degradation under different environmental conditions.
The bacteria were isolated based on viscosity reduction of the xanthan solution. Bacterial isolates were identified using rep-PCR (repetitive element-based genomic fingerprinting) and 16S rDNA sequencing. Xanthanases were characterized by measuring their activity at different temperatures, pH values, and NaCl concentrations. Degradation of other polysaccharides and xanthan degradation products were investigated based on the screening plate method and TLC (thin-layer chromatography), respectively.
Six isolates from different Paenibacillus species with a complete xanthan degrading capability were isolated from Urmia Lake. Phylogenetic analysis placed these strains within the genus Paenibacillus with the closest relatives that were found to be P. nanensis, P. phyllosphaerae, P. agaridevorans, P. agarexedens, and P. taohuashanense. These isolates displayed different levels of the xanthan biodegradation activity in temperatures ranging from 15 to 55 °C and pH values from 4 to 11. Xanthanolytic activity was generally prevented in presence of NaCl (> 0.1 mol.L-1). Furthermore, the isolated Paenibacillus spp. could degrade several other polysaccharides including xylan, CMC (carboxymethyl cellulose), starch, alginate, and pectin.
Novel strains of the six different Paenibacillus species that were introduced in the present study are able to produce xanthanases with interesting characteristics. In light of the results from this study, special applications, particularly in healthcare, medicine, and the environment is hereby proposed for these enzymes.

Pseudomonas aeruginosa biofilm is of interest due to its involvement cystic fibrosis and prosthesis-related infections. Unique biofilm structure, the vast application of antibiotics and accordingly antibiotic resistance, increase the need for new drug delivery methods to enhance drug efficiency. Nanocarriers are one of the new choices. The aim of the presented study is to evaluate the efficiency of amikacin-loaded PLGA nanoparticles in P. aeruginosa biofilm eradication.
Antibacterial (MIC and MBC) and antibiofilm (MBEC) activity of synthesized nanoparticle, by double emulsification method, was assayed against P. aeruginosa PAO1.
The synthesized nanoparticles contain an appropriate amount of antibiotic )≈26 μg Amikacin/mg/ nanoparticle ) and have a suitable size )≈440 nm) for inhalation and injection. The particles are sustainable, have a good antibiotic release kinetic for antibacterial applications. Furthurmore, the antibiotic loaded in the particles also keep their antibacterial activity.
Amikacin is known to possess nephrotoxic and ototoxic properties and therefore loading it in a polymeric cover, makes the nanoparticles suitable candidate antibacterial and antibiofilm applications with fewer side effects.

Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR). Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol.
According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications.
This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s) of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.
Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

The USEPA has suggested faecal enterococci as the primary bacterial indicators. Of more importance is their direct correlation with swimmer-associated gastroenteritis in recreation water quality monitoring. In contrast to other seawater bodies with 3.5% salinity, the recreational waters in the southern coast of the Caspian Sea possess its own salinity (about 1% w/v) and thus require further investigations to determine the capacity of Enterococcus faecalis as the sole primary microbial index in this unique aquatic environment.
The survey of the presence and survival of E. faecalis as a microbial index in the recreational waters of the southern Caspian Sea was carried out using a microcosm as an experimental model. The concentration of E. faecalis cells in samples of seawater were estimated by a standard membrane filtration method using m-Enterococcus agar as the selective culture medium. As the current standard culture-based methods are not reliable enough for the detection of non-growing, damaged and under-tension bacteria, PCR was used to identify the possible VBNC form of the bacterium after disappearing of the culturable cells.
Results and A continuous decline in the number of culturable E. faecalis cells resulted in apparent elimination of the bacteria from seawater in a defined period. Detection of intact DNA was possible in the following 60 days. The salinity of about 1% and the self-purification properties of the Caspian Sea make the conditions feasible for the use of this microorganism as a measure of water quality throughout the region. The results confirmed the presence of damaged bacterial cells, namely VBNC forms, indicating the necessity of examining of the sea water samples by using molecular approaches or repair procedures.

Low grade grape juice concentrate was used as carbon source for xanthan production. Significant factors affecting xanthan concentration, productivity and viscosity were investigated using Plackett-Burman Design. Based on the obtained results, carbon and nitrogen concentrations, inoculum size and agitation rate, were assumed as significant factors. Broth culture viscosity and xanthan concentration were optimized using Response Surface Methodology with four independent variables: carbon source (30, 40, 50 g l-1), ammonium sulfate as nitrogen source (0.5, 1.25, 2 g l-1), agitation (150, 200, 250 rpm) and inoculum size (5, 10, 15% v v-1). Optimum level for each factor was obtained by desirability function approach. The average of xanthan gum production and its viscosity under optimized conditions were recorded as 14.35 g l-1 and 1268 cP, respectively. The average yield of production and productivity of xanthan within 72 h under optimized conditions were 35% and 0.19 g l-1 h-1, respectively. The current study showed the potential of low-grade grape juice concentrate as an economic carbon source for xanthan gum production.

Microbial analysis of ground water, as the sole supplying water source in many areas, must be evaluated. Because the treatment of water cannot remove all pathogenic bacteria leaked from domestic wastewater, bacterial analysis of Bojnourd groundwater sources was performed. For this reason, membrane filter (MF) technique and Most Probable Number (MPN) method were used to evaluate the microbial quality of the water. Escherichia coli (E. coli) and Enteroccocus faecalis (E. faecalis) were traced as excremental indices. E. coli was detected from three out of six stations and E. faecalis was only isolated from one station. Although molecular techniques are very rapid and exact methods for detection of microbial community and can identify ‘Viable But Not Cultivable’ (VBNC) bacteria, they are unable to make a distinction between living and non-living microorganisms. By means of a standard technique, it is possible to study living and metabolically active microorganisms. Due to the detection of E.coli and E.feacalis in some stations the sanitization of groundwater must be revised to lessen the microbial population in this groundwater.

Mixed aerobic and anoxic conditions، in addition to substrate versatility، provide a wide range of microhabitats and niches for a large number of microbial species including bacterian yeasts and filamentous fungi. Among them، study of environmental yeast species، especially those producing secretory catabolic enzymes، can be of a great importance. After enrichment of water and soil samples and their cultivation on antibiotic supplemented YPG Agar، 46 isolates were obtained. Most of the isolates were capable of producing nucleases and sterases. Using cup plate method revealed that strains SA044 and SA006 were able to produce the greatest number of secretory enzymes. Production of at least one secretory amylase، protease، nuclease and lipase was recorded in one or both of these isolates. Production of secretory keratinase، cellulose، phytase، xylanase، chitinase and pectinase was not observed. Using molecular approach and phenotypic examinations، the isolates SA044 and SA006 were identified as Pseudozyma aphidis and Pseudozyma antarctica respectively.

The genus Xanthomonas is composed of phytopathogenic bacterial species. In addition to causing crops diseases, most of the Xanthomonas species especially Xanthomonas campestris produce xanthan gum via an aerobic fermentation process. Xanthan gum is, an important exopolysaccharide from Xanthomonas campestris, mainly used in the food, petroleum and other industries. the purpose of this study was assessment of relationship between genetic diversity and xanthan production in Xanthomonas spp. In this study 15 strains of Xanthomonas spp. which had previously been isolated from soils of vegetable farms, were discriminated from each other using Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and 16S rDNA sequencing methods. Xanthan production of strains was measured in 250 ml flask. The results of ERIC PCR and xanthan production was compared. ERIC-PCR patterns not only could differentiate all Xanthomonas campestis from the control i.e. Xanthomonas translucent but also discriminate strains of Xanthomonas to three clusters with 40% similarity based on Jaccard’s coefficient. This clustering of the strains was in agreement with other characteristics including xanthan production and biochemical features. The results showed that genomic fingerprinting conferred adequate genetic data for discriminating between strains of the species Xanthomonas campestris. The data indicated a partial relationship between ERIC-PCR patterns and xanthan production by the strains. Further development of experiments may result in making good prediction about xanthan production capabilitymof the Xanthomonas strains on the basis of ERIC-PCR method.

Rhodotorula is characterized by the absence of ballistoconidia, fermentation ability, and starch-like compounds. Biology of the species is not well-identified; Therefore molecular identification is required. Sequence analysis of the D1/D2 region can be used for the identification of the majority of Basidiomycetous species. Carotenoids which are natural pigments can be synthesized by some genera of yeasts such as Rhodotorula. The increase of demand for carotenoids obtained from natural sources has promoted major efforts to recognize potential microbial sources. The aims of this study were to identify a strain isolated from leather wastewater and to investigate its carotenoid production ability. The effect of 2 different medium (Semi-synthetic medium (MMS) and Yeast Malt extract medium (YM)) on biomass and carotenoid production was studied. In this experimental study, Sequence analysis of the D1/D2 region in addition to morphological and biochemical characterization to identify the strain was carried out. To isolate the carotenoid pigment, cells were suspended in acetone and broken using a homogenizer, followed by centrifugation and supernatant was separated; Thus pigments were measured spectrophotometrically at 450 nm using the extinction coefficient E1%450=2500. Identification processes represented strain SG006 as a Rhodotorula slooffiae. The sequence was deposited in the Gene Bank database with accession number JX997835. The results showed that SG006 are able to produce carotenoid and MMS medium promoted carotenoid production. We found that Rhodotorula slooffiae showed the ability to produce carotenoid. However, further work is needed to optimize of the amount of product and to characterize the carotenoids.

Although bacteria and molds are the pioneering microorganisms for production of many enzymes, yet yeasts provide safe and reliable sources of enzymes with applications in food and feed. Single xylanase producer yeast was isolated from plant residues based on formation of transparent halo zones on xylan agar plates. The isolate showed much greater endo-1, 4-β-xylanase activity of 2.73 IU/ml after optimization of the initial extrinsic conditions. It was shown that the strain was also able to produce β-xylosidase (0.179 IU/ml) and α-arabinofuranosidase (0.063 IU/ml). Identification of the isolate was carried out and the endo-1, 4-β-xylanaseproduction by feeding the yeast cells on agro-industrial residues was optimized using one factor at a time approach. The enzyme producer strain was identified as Aureobasidiumpullulans. Based on the optimization approach, an incubation time of 48 hr at 27°C, inoculum size of 2% (v/v), initial pH value of 4 and agitation rate of 90 rpm were found to be the optimal conditions for achieving maximum yield of the enzyme. Xylan, containing agricultural residues, was evaluated as low-cost alternative carbon source for production of xylanolytic enzymes. The production of xylanase enzyme in media containing wheat bran as the sole carbon source was very similar to that of the medium containing pure beechwoodxylan. This finding indicates the feasibility of growing of A. pullulans strain SN090 on wheat bran as an alternate economical substrate in order for reducing the costs of enzyme production and using this fortified agro-industrial byproduct in formulation of animal feed.

Synthetic dyes are recalcitrant to degradation and toxic to different organisms. Decolorization of textile wastewaters is one of the major concerns since last decades. Physical-chemical treatments are very expensive and frequently producing large amounts of toxic wastes. Biological treatments can be more convenient. In the present study, an attempt has been made for decolorization of azo dyes using microbial process. Screening of microorganisms capable of azo dye decolorization was performed from activated sludge. The decolorization of various dyes (Reactive Black 5, Reactive Orange 16, Reactive Red 198, Direct Blue 71, Direct Yellow 12 and Direct Black 22) was determined by measuring the absorbance of culture supernatant at their λmax. Culture supernatants were also analyzed for UV-Vis absorption between 200-800 nm. The effect of aeration, temperature, different concentrations of glucose and NaCl was studied with an aim to determine the optimal conditions required for maximum decolorization. The yeast (strain JKS4) which had high ability to decolorize different azo dyes was isolated. Under aerobic condition, the yeast strain showed 85.7% of decolorization at 200 mg/l Reactive Black 5 (as a model azo dye), 1% (w/v) glucose concentration and 35°C after 24 h. All the examined dyes were extensively decolorized (53.35-97.9%) after 24 h. With elongated incubation period, complete decolorization was observed in presence of all dyes. From the physiological properties and phylogenetic analysis based on the 26S rDNA sequences, strain JKS4 was classified into Candida palmioleophila. Because of high decolorizing activity against various azo dyes commonly used in the textile industries, it is proposed that the isolated yeast may have a practical application in the biotransformation of various dye effluents.

Carotenoids which are naturally synthesized by fungi such as yeasts can act as an antioxidant which is closely related to their ability to decrease the risk of a variety of degenerative diseases. In recent years, the increase of demand for carotenoids obtained from natural sources has promoted major efforts to improve carotenoid production from biological sources such as pigmented yeasts. The aim of this study was comparing incubation time and carotenoid production in Rhodotorula slooffiae and R. mucilaginosa isolated from leather tanning wastewater. To isolate the carotenoid pigment, cells were suspended in acetone and broken using a homogenizer, followed by centrifugation and separation of supernatant. In order to study the effect of incubation time, samples were held at 30 С in a shaker at 150 rpm for 24, 48, 72, 96, and 120 hr. For analytical evaluation, pigments were measured spectrophotometrically at 450 nm using the extinction coefficient E1%450=2500. The results showed that the content of total carotenoid in R. slooffiae was the highest when samples were incubated for 72 hr. Overall, R. mucilaginosa had more potential to produce carotenoid. The best incubation periods for R. slooffiae and R. mucilaginosa were 72 hr and 48 hr, respectively. It seemed that the maximum rate of total carotenoid was not directly associated with the maximum amount of cell biomass and the type of carotenoid and their relative amount may vary depending on genus of yeast.

Aqueous extract of dried fourth stomach of unweaned bovine contains prochymosin, that subsequently is converted to active chymosin, a milk-coagulating enzyme in the cheese industry. Recombinant chymosin has been found to be a suitable replacement for natural bovine chymosin. Meanwhile it possesses several advantages to other plant, fungal and bacterial milk-clotting enzymes.. In present research we evaluate the expression of this critical enzyme in a eukaryotic system for future use in cheese industry.. We have cloned bovine prochymosin gene in methylotrophic yeast, P. pastoris, using pPIC9K as an expression vector. The recombinant plasmid was transformed into the host by electroporation, and it was expressed in optimum conditions (temperature 29oC, 200 rpm, 2% methanol for induction, and 5 days of incubation). Transcription and expression of the recombinant prochymosin was evaluated by the reverse transcription polymerase chain reaction (RT-PCR), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis as well as western blotting and enzyme-linked immunosorbent assay (ELISA).. In optimum conditions, only a low level of this heterologous protein was detected using ELISA method and subsequently confirmed by RT-PCR.. Since it has been reported that P. pastoris is an appropriate host for the expression of recombinant proteins, a low level of expression of prochymosin in this host should be explored in our future research..