Integrating bioprocess and metagenomics studies to enhance humic acid production from rice straw.

Rice straw burning annually (millions of tons) leads to greenhouse gas emissions, and an alternative solution is producing humic acid with high added-value. This study aimed to examine the influence of a microbial consortium and other additives (chicken manure, urea, olive mill waste, zeolite, and biochar) on the composting process of rice straw and the subsequent production of humic acid. Results showed that among the fungal species, Thermoascus aurantiacus exhibited the most prominent impact in expediting maturation and improving compost quality, and Bacillus subtilis was the most abundant bacterial species based on metagenomics analysis. The highest temperature, C/N ratio reduction, and amount of humic acid production (Respectively in lab 61 °C, 54.67%, 298 g kg-1 and in pilot level 65 °C, 72.11%, 310 g kg-1) were related to treatments containing these microorganisms and other additives except urea. Consequently, T. aurantiacus and B. subtilis can be employed on an industrial scale as compost additives to further elevate quality. Functional analysis showed that the bacterial enzymes in the treatments had the highest metabolic activities, including carbohydrate and amino acid metabolism compared to the control. The maximum enzymatic activities were in the thermophilic phase in treatments which were significantly higher than that in the control. The research emphasizes the importance of identifying and incorporating enzymatically active strains that are suitable for temperature conditions, alongside the native strains in decomposing materials. This strategy significantly improves the composting process and yields high-quality humic acid during the thermophilic phase.

Native chicken-derived Lactobacillus spp. strains with high probiotic, cholesterol-assimilation and aflatoxin-degradation capabilities.

Background and Objectives: Probiotics are added into the food or feed systems and provide beneficial effects to the human or animal health. This study aimed to isolate the gastrointestinal native Lactobacillus strains with high probiotic, cholesterol-assimilation and aflatoxin-degradation capabilities from native chickens. Materials and Methods: About 70 Lactobacillus isolates were isolated from ileum of the Fars province native chickens and were investigated for their probiotic properties. Results: Of 70 Lactobacillus isolates, 10 showed high probiotic capabilities, including survival at acidic conditions (pH up to 2.5), tolerance of 0.5% bile and 6-10% NaCl salts, growth in a wide range of temperature from 15 to 45°C, antagonistic effects against different important bacterial pathogens (Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, Clostridium defficile, Enterococcus hirae, Salmonella enterica and Staphylococcus aureus) and sensitivity to some important antibiotics. The selected strains had an aggregation time less than 120 min. The 16S-rDNA sequencing showed that the selected strains were highly related to Lactobacillus reuteri and L. casei. Finally, the selected strains in this study along with 10 other probiotic strains isolated and characterized in our pervious study were used to evaluate their cholesterol assimilation and aflatoxin B1 degradation capabilities. The potentials of cholesterol assimilation of the selected strains were significantly different (P<0.05) and ranged from 2.3% to 99%. The highest content of cholesterol assimilation was obtained in isolates M20 and M4 with more than 98% absorption. Moreover, four strains 43, OR7, M21 and OR9 were able to absorb AFB1 with 58.6%, 52.33%, 47% and 31.6% efficiency respectively. Conclusion: It could be concluded that the strains 43, M21 and OR7 showed high probiotic potentials for application in the poultry industry.

Distribution of Soil Extracellular Enzymatic, Microbial, and Biological Functions in the C and N-Cycle Pathways Along a Forest Altitudinal Gradient.

The diverse chemical, biological, and microbial properties of litter and organic matter (OM) in forest soil along an altitudinal gradient are potentially important for nutrient cycling. In the present study, we sought to evaluate soil chemical, biological, microbial, and enzymatic characteristics at four altitude levels (0, 500, 1,000, and 1,500 m) in northern Iran to characterize nutrient cycling in forest soils. The results showed that carbon (C) and nitrogen (N) turnover changed with altitude along with microbial properties and enzyme activity. At the lowest altitude with mixed forest and no beech trees, the higher content of N in litter and soil, higher pH and microbial biomass nitrogen (MBN), and the greater activities of aminopeptidases affected soil N cycling. At elevations above 1,000 m, where beech is the dominant tree species, the higher activities of cellobiohydrolase, arylsulfatase, ß-xylosidase, ß-galactosidase, endoglucanase, endoxylanase, and manganese peroxidase (MnP) coincided with higher basal respiration (BR), substrate-induced respiration (SIR), and microbial biomass carbon (MBC) and thus favored conditions for microbial entropy and C turnover. The low N content and high C/N ratio at 500-m altitude were associated with the lowest microbial and enzyme activities. Our results support the view that the plain forest with mixed trees (without beech) had higher litter quality and soil fertility, while forest dominated by beech trees had the potential to store higher C and can potentially better mitigate global warming.

Whole genome sequence of Pantoea agglomerans ANP8, a salinity and drought stress-resistant bacterium isolated from alfalfa (Medicago sativa L.) root nodules.

Environmental abiotic stress conditions, especially drought and salinity, are currently the major factors that reduce crop yields worldwide. It has been reported that plant-associated beneficial bacteria, especially strains resistant to abiotic stresses that could maintain their efficiency under environmental challenging conditions, can contribute to alleviate abiotic stresses of host plants. In this study, we presented the assembly of the whole genome of Pantoea agglomerans ANP8, a plant growth-promoting bacterium resistant to salinity and drought stresses. The draft genome assembly contained 4,713,172 bp with 4586 predicted genes. A primary draft genome with a total of 5,115,548 bp and 1916 contigs was obtained (longest contig length being 485,272 bp and smallest contig being 112 bp). Following assembly upgrades, 68 scaffolds and 70 contigs with lengths ≥ 500 bp and an N50 = 209,657 bp were obtained. Number of 5554 and 5472 open reading frames longer than 50 codons were observed in the direct strand and in the reverse strand, respectively. Due to the multiple plant growth-promoting characteristics of this bacterium, genes involved in various indole-3-acetic acid production pathways, e.g., indole-3-pyruvic acid and indole-3-acetamide pathways, were found in the bacterium's genome. In addition, multiple copies of the gcd gene, most important enzymes involvement in the solubilization of phosphates, glucose dehydrogenase, were also observed in this genome. The study provides new genomic information to help understanding the way of action of a stress-tolerant plant growth-promoting bacterium.

Composition of soil bacterial and fungal communities in relation to vegetation composition and soil characteristics along an altitudinal gradient.

The objective of the present study was to evaluate how altitudinal gradients shape the composition of soil bacterial and fungal communities, humus forms and soil properties across six altitude levels in Hyrcanian forests. Soil microbiomes were characterized by sequencing amplicons of selected molecular markers. Soil chemistry and plant mycorrhizal type were the two dominant factors explaining variations in bacterial and fungal diversity, respectively. The lowest altitude level had more favorable conditions for the formation of mull humus and exhibited higher N and Ca contents. These conditions were also associated with a higher proportion of Betaproteobacteria, Acidimicrobia, Acidobacteria and Nitrospirae. Low soil and forest floor quality as well as lower bacterial and fungal diversity characterized higher altitude levels, along with a high proportion of shared bacterial (Thermoleophilia, Actinobacteria and Bacilli) and fungal (Eurotiomycetes and Mortierellomycota) taxa. Beech-dominated sites showed moderate soil quality and high bacterial (Alphaproteobacteria, Acidobacteria, Planctomycetes and Bacteroidetes) and fungal (Basidiomycota) diversity. Particularly, the Basidiomycota were well represented in pure beech forests at an altitude of 1500 m. In fertile and nitrogen rich soils with neutral pH, soil quality decreased along the altitudinal gradient, indicating that microbial diversity and forest floor decomposition were likely constrained by climatic conditions.

Comparative Analyses of Transport Proteins Encoded within the Genomes of Bdellovibrio bacteriovorus HD100 and Bdellovibrio exovorus JSS.

Bdellovibrio, δ-proteobacteria, including B. bacteriovorus (Bba) and B. exovorus (Bex), are obligate predators of other Gram-negative bacteria. While Bba grows in the periplasm of the prey cell, Bex grows externally. We have analyzed and compared the transport proteins of these 2 organisms based on the current contents of the Transporter Classification Database (TCDB; www.tcdb.org). Bba has 103 transporters more than Bex, 50% more secondary carriers, and 3 times as many MFS carriers. Bba has far more metabolite transporters than Bex as expected from its larger genome, but there are 2 times more carbohydrate uptake and drug efflux systems, and 3 times more lipid transporters. Bba also has polyamine and carboxylate transporters lacking in Bex. Bba has more than twice as many members of the Mot-Exb family of energizers, but both may have energizers for gliding motility. They use entirely different types of systems for iron acquisition. Both contain unexpectedly large numbers of pseudogenes and incomplete systems, suggesting that they are undergoing genome size reduction. Interestingly, all 5 outer-membrane receptors in Bba are lacking in Bex. The 2 organisms have similar numbers and types of peptide and amino acid uptake systems as well as protein and carbohydrate secretion systems. The differences observed correlate with and may account, in part, for the different lifestyles of these 2 bacterial predators.

Characterization of some potentially probiotic Lactobacillus strains isolated from Iranian native chickens.

The objective of the present study was to isolate, identify and characterize new LAB strains with high probiotic potentials from Iranian (Isfahan) indigenous chickens. From 90 isolated LABs, 11 isolates had high growth rate under different stress conditions, including acid (pH 2.5), bile (0.5% oxgall), salt (6-15%) and temperatures 15 and 45°C, and their aggregation time was less than 120 min. Based on the molecular identification using 16S rDNA sequencing and phylogenetic analysis, the isolates belonged to two Lactobacillus salivarius and L. reuteri species. The isolates showed different tolerance to 16 clinically and veterinary relevant antibiotics, and most of them were resistant to or semi-tolerant of 7-15 different studied antibiotics. The Es11, Es12, Es3 and Es13 strains with resistance to or semi-tolerance of 15, 14 and 13 different antibiotics, respectively, were the most tolerant strains. The selected isolates showed a wide range of antimicrobial activity against 7 different pathogenic strains. All the isolates exhibited antagonistic activity against E. coli, Enterococcus hirae, Salmonella enterica and Staphylococcus aureus. The isolates Es6 and Es11 with high antagonistic activity and resistance against 6 of the studied pathogens were the most powerful antagonistic isolates. The values and types of adhesion to the Caco-2 cell cultures were significantly different (0-40 bacteria/Caco-2 cell), and the maximum adhesion was observed for the isolates Es6 and Es13 with 35 and 40 bacteria adhesion/cell, respectively. Finally, based on all the experiments, 7 strains, including Es1, Es6, Es7, Es11, Es12 and Es13, were selected for the further in vivo assays and possible use in the poultry industry.

Impact of host plant resistance on the tritrophic interactions between wheat genotypes, Schizaphis graminum (Homoptera: Aphididae), and Coccinella septempunctata (Coleoptera: Coccinellidae) using molecular methods.

The objective of the current study was to evaluate the impact of wheat resistance on digestibility of Schizaphis graminum (Rondani) in the Coccinella septempunctata L. gut. Four wheat genotypes including an ancient wheat species (Einkorn) and three Iranian wheat genotypes (Azadi, Ommid, and Moghan2) were used. Einkorn was found to be a highly resistant species, while Azadi, Ommid, and Moghan2 were characterized as resistant, semiresistant, and susceptible genotypes, respectively, based on some biological parameters of S. graminum on these plants. Adult C. septempunctata starved for 24 h were fed one S. graminum reared on the selected wheat genotypes. A semiquantitative polymerase chain reaction technique using specific primers for the gene cytochrome oxidase II (COII) of S. graminum showed a significant effect of plant resistance on S. graminum DNA disappearance time. The DNA half-lives of S. graminum fed on Einkorn, Azadi, Ommid, and Moghan2 in C. septempunctata gut was 3.3, 3.6, 4.8, and 6.7 h, respectively. The findings suggest that digestibility of S. graminum fed on resistant plant genotypes is higher than those fed on susceptible plants because of the lighter weights and smaller sizes of the first. This phenomenon could result in faster ingestion of S. graminum by C. septempunctata on these resistant plants. It is also possible that some metabolites found differentially in Einkorn and Azadi genotypes may have affected S. graminum quality as a food for C. septempunctata.

Draft genome sequence of Ureibacillus thermosphaericus strain thermo-BF, isolated from Ramsar hot springs in Iran.

Ureibacillus thermosphaericus strain Thermo-BF is an aerobic, thermophilic bacillus which has been characterized to biosynthesize gold nanoparticles. Here we present the draft genome sequence of Ureibacillus thermosphaericus strain Thermo-BF which consists of a 2,864,162-bp chromosome. This is the first report of a shotgun sequenced draft genome of a species in the Ureibacillus genus.

Evaluation of anti-phytoplasma properties of surfactin and tetracycline towards lime witches' broom disease using real-time PCR.

The anti-phytoplasma activities of surfactin (derived from Iranian native Bacillus subtilis isolates) and tetracycline towards Candidatus "Phytoplasma aurantifolia", the agent of lime Witches' broom disease, were investigated. HPLC was used to quantify the surfactin production in four previously characterized native surfactin-producing strains, and the one producing the highest amount of surfactin (about 1,500 mg/l) was selected and cultivated following optimized production and extraction protocols. Different combinations of purified surfactin and commercial tetracycline were injected into artificially phytoplasmainfected Mexican lime seedlings using a syringe injection system. An absolute quantitative real-time PCR system was developed to monitor the phytoplasma population shifts in the lime phloem during 3 months following the injections. The results revealed that the injections of surfactin or tetracycline had a significant inhibitory effect on Candidatus "P. aurantifolia". However, the combined treatment with both surfactin and tetracycline (1:1) resulted in the highest inhibition due to a synergic effect, which suppressed the phytoplasma population from about 2×10(5) to less than 10 phytoplasma units/g plant tissue.

Molecular and biochemical characterization of Iranian surfactin-producing Bacillus subtilis isolates and evaluation of their biocontrol potential against Aspergillus flavus and Colletotrichum gloeosporioides.

The characterization of surfactin-producing Bacillus subtilis isolates collected from different ecological zones of Iran is presented. Characterization was performed using blood agar, PCR, drop-collapse, and reverse-phase high-performance liquid chromatography (HPLC) analyses, and the isolates' biocontrol effects against the aflatoxin-producing agent Aspergillus flavus and the citrus antracnosis agent Colletotrichum gloeosporioides were studied. In total, 290 B. subtilis isolates were isolated from phylosphere and rhizosphere samples collected from fields and gardens of 5 provinces of Iran. Blood agar assays showed that 185 isolates produced different biosurfactants. Isolates containing the sfp gene, coding for surfactin, were detected using the PCR method. It was found that 14 different isolates contained the sfp gene. Drop-collapse assays, which detect isolates with high production of surfactin, showed that 7 isolates produced high levels of surfactin. It was found from HPLC analysis that the isolates containin the sfp gene produced between 55 and 1610 mg of surfactin per litre of broth medium. Four isolates, named BS119m, BS116l, N3dn, and BS113c, produced more than 1000 mg of surfactin per litre of broth. The highest surfactin production level was observed for isolate BS119m (1610 mg/L). The antagonistic potential of the sfp gene-containing isolates was determined using dual culture and chloroform vapour methods. Our bioassay results indicated that isolate BS119m showed high inhibitory effects against A. flavus (100%) and C. gloeosporioides (88%). Furthermore, the effect of purified surfactin on the growth of A. flavus was evaluated. Mycelia growth was considerably reduced with increasing concentration of surfactin, and 36%, 54%, 84%, and 100% inhibitions of mycelia growth were, respectively, observed at 20, 40, 80, and 160 mg/L after 7 days of incubation.

Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes.

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.